Session V50: Physics of Development and Disease - II

Visualizing traumatic brain injury in vivo

Recent advances in imaging technology have made it possible to visualize neuroimmune communication in the living brain. We have recently begun to unravel the mechanisms underlying innate immune cell dynamics in the brain and meninges during states of health and disease. This lecture will focus on how CNS resident myeloid cells coordinate with peripherally-derived myelomonocytic cells to clear dead cells and rebuild damaged blood vessels following traumatic brain injury (TBI). A special emphasis will be placed on the use of intravital two-photon laser scanning microscopy to study how the brain responds in real-time to TBI.   

Human Brain Organoids on a Chip Reveal the Physics of Wrinkling

The origin of human brain wrinkling remains an open fundamental problem, with implications to neurodevelopmental disorders. Studies in polymer gel models suggest that wrinkling emerge spontaneously due to the development of compression forces during differential swelling, however these ideas have not been tested in a living system. Here, we report the appearance of surface wrinkles during in vitro development and self-organization of human brain organoids, in a micro-fabricated compartment, which supports in situ imaging over weeks. By studying the cellular dynamics, we observed nuclear nematic ordering and compression during development. Convolutions emerged at a critical nuclear density, which is indicative of a mechanical instability. We identified two opposing forces which contribute to differential growth; cytoskeletal contraction at the organoid core, and nuclear expansion during cell-cycle at the organoid perimeter. The wrinkling wavelength exhibited linear scaling with tissue thickness, consistent with an equilibrium between bending and stretching energies. Finally, lissencephalic (smooth brain) organoids displayed reduced convulsions, linear scaling with an increased prefactor, and reduced elastic modulus.   

Effects of maturation factors on the mechanics and electrophysiology of 3D engineered cardiac microtissues

Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) represent a potentially unlimited source of cells that can be used for cardiac regeneration and cardiac disease modeling. However, hiPSC-CMs as currently used remain fundamentally immature. We have developed three-dimensional (3D) engineered cardiac microtissues (CMTs) grown on magnetically actuated microfabricated tissue gauges, which enable assessment of the physical and electrophysiological characteristics of the hiPSC-CMs through measurements of their force generation and mechanical response to electrical and chemical perturbations. Using combinations of defined chemical factors including triiodothyronine hormone, we have demonstrated structural maturation of hiPSC-CMs in CMTs, including improved alignment and well-defined sarcomeres more similar to adult CMs than untreated cells. The treated CMTs show enhanced static and dynamic force generation, and increased spontaneous contraction frequency. Measurements of conduction velocity via voltage-sensitive optical mapping show that the CMTs are well coupled electrically. These 3D hiPSC-CMTs can serve as a platform for exploration of the physical manifestations of genetically based cardiac diseases.
  

Epithelialization at Forming Somite Boundaries

Mesenchymal to epithelial transition is an emergent mechanical process which results in mesenchymal cells attaining structure and polarity along with the formation of basal and apical protein domains. We study this transition in the context of somite formation which is an important part in the development of vertebrate embryos. Randomly distributed mesenchymal cells in the continuous presomitic mesoderm organize into strongly coupled epithelial cells forming a monolayer in a somite. Data from chicken embryos show epithelialization along the regions close to the ectoderm and endoderm first, followed by closing of the somite anteriorly and posteriorly. Here we explore what cell mechanics guide the emergent process of epithelialization at these boundaries. We model the transition from undifferentiated mesenchyme to elongated epithelia using filopodial processes and show that propagation of epithelial order and accretion \footnote{ H.Y.Kim,et al, \emph{On the role of mechanics in driving mesenchymal-to-epithelial transitions}, Semin Cell Dev Biol, (2016)} is sufficient to explain the observed monolayer. We further show how subsequent apical constriction of the formed layer explains the rounding of somite boundaries.   

Electric Impedance Monitoring of Cell Migration Following Irradiation

Cell migration is a crucial step in cancer metastasis, the complex process which accounts for over 90% of cancer-related deaths. There is emerging clinical evidence that radiotherapeutic doses meant to kill cancer cells can promote metastasis by enhancing cell migration. Here we quantify radiation-induced changes in the migration of three different cancer cell lines in order to ascertain the lineage-dependence of the pro-metastatic effect of radiation.
We subjected K562 (erythroid), HL60 (myeloid) and HCN2 (neuronal) cancer cells to low dose and high dose ionizing radiation by means of a radiotherapy-grade cell irradiator (CellRad, Faxitron). We used a commercial impedance sensing device (ECIS®) to assess the migration of the cells following irradiation.
The migration all three cell lines in ECIS, was significantly altered by the X-rays. We will present results comparing the pro-metastatic effects of X-rays on the three cell lines as well as evidence of cytoskeletal alterations behind the measured changes in migration, potentially providing a new rationale for the improvement of radiotherapy protocols and the search for anti-metastasis drugs.
  

Mechanism for Amplitude Alternans in Action Potential and the Initiation of Spatiotemporal Chaos in the Heart.

It is widely believed that one major life-threatening transition to chaotic fibrillation occurs via spiral-wave breakup that is preceded by spatiotemporal dispersion of refractoriness due to alternations in the duration of the cardiac action potential (AP). However, recent clinical and experimental evidence suggests that other characteristics of the AP may contribute to, and perhaps drive, this dangerous dynamical instability. To identify the relative roles of AP characteristics, we performed experiments in rabbit hearts under conditions to minimize AP duration dynamics which unmasked pronounced AP amplitude alternans just before the onset of fibrillation. We used a simplified ionic cell model to derive a return map and a stability condition that elucidates a novel underlying mechanism for AP alternans and spiral breakup. We found that inactivation of the sodium current is key to developing amplitude alternans and is directly connected to conduction block and initiation of arrhythmias. Simulations in 2D where AP amplitude alternation led to turbulence confirm our hypothesis.   

The non-uniform charge distribution along the N-terminal domain of virus coat proteins modifies the stability of virus icosahedral shells.

Many spherical viruses encapsulate their genome in protein shells with icosahedral symmetry. This process is spontaneous and driven by electrostatic interactions between positive domains on the virus coat proteins and the negative genome. We study the effect of the non-uniform charge distribution along the N-terminal domain of virus coat proteins and icosahedral charge distribution of the protein shell using a mean-field theory. Our goal is to study the impact of non-uniform charge distribution on the free energy and stability of viral particles.   

Quantitative Electrophysiological Monitoring of Anti-histamine Drug Effects on Live Cells via Reusable Sensor Platforms

We report a method for the quantitative electrophysiological monitoring of histamine and anti−histamine drug effects on live cells via reusable sensor platforms based on carbon nanotube transistors. This method enabled us to repeatedly measure the real−time electrophysiological responses of a single HeLa cell to histamine with different concentrations. The measured electrophysiological responses were attributed to the activity of histamine type 1 receptors on a HeLa cell membrane by histamine. Furthermore, the effects of anti−histamine drugs such as cetirizine or chlorphenamine on the electrophysiological activities of HeLa cells were also evaluated quantitatively. Significantly, we utilized only a single device to monitor the responses of multiple HeLa cells to each drug, which allowed us to quantitatively analyze the antihistamine drug effects on live cells without suffering from errors by the device-to-device variations of device characteristics. Since our method takes the statistically-meaningful quantitative evaluation capability, this would promise versatile applications for drug screening and nanoscale bio sensor researches.   

Agent-based Model for Developmental Aggregation in Myxococcus xanthus Bacteria

Spatial self-organization is widely studied in active matter physics due to its biological significance. Myxococcus xantus is a rod-shaped soil bacterium that can serve as a simple model system to study the self-organization because under different conditions, its cells self-organize into distinct dynamical patterns. To understand how M. xantus cells aggregate into multicellular mounds under starvation conditions, we built an agent-based model. In this model, each cell is modeled as an agent, represented by a point-particle and characterized by its position and moving direction. At low agent density, the model recapitulates the dynamic patterns observed by experiments. However, at high agent density, this model results in formation of cell streams but not stable aggregates. To overcome this problem, we extend the model based on the recent experimental observation that cells tend to have a longer run period when moving towards aggregate. We assumed that cells produce a chemical signal that affects their reversal frequency. Using a phenomenological chemotaxis model with adaptation, we can match run duration bias with observed experimental results. Incorporating these effects in our model leads to the formation of stable aggregates.   

Extinction and Speciation in Red Queen's Race of Influenza Virus.

The success of influenza as a virus is due to its ability to escape the human immune system memory by rapidly mutating its antigens. The continuous arms race between the virus and the adaptive immunity of its host population drives the need to update flu vaccines every few years. It also shapes a peculiar, spindly phylogenic-tree structure of influenza A (H3N2), with a single continuous backbone going forward accumulating antigenic innovations, while overall viral diversity at any given time remains limited. Thus, H3N2 flu evolves fast enough to reliably evade host immunity, yet it does not accumulate diversity, leading to divergence and eventual "speciation" in the sense of the loss of cross-immunity imparted by coexisting flu genotypes. What maintains this delicate balance? Under what conditions is the flu destined to extinction or speciation? From an epidemic-type model capturing this Red Queen's race between virus and host-immunity, we identify the time delayed (and nonlinear) feedback that stabilizes viral population (over a finite range of evolutionary parameters) so that the subspecies cloud moves as a traveling wave in the antigenic space, giving rise to a persistent but un-branching phylogenic backbone.   

The Vibrio cholerae Type VI Secretion System Can Modulate Host Intestinal Mechanics to Displace Commensal Gut Bacteria

Host-associated microbiota help defend against bacterial pathogens; the physical and chemical mechanisms by which pathogens overcome this defense, however, remain largely unknown. We applied live imaging to larval zebrafish to study how the human pathogen Vibrio cholerae invades the intestine. The gut microbiota of fish mono-colonized by commensal strain Aeromonas veronii was displaced by V. cholerae expressing its Type VI Secretion System (T6SS), a syringe-like apparatus that deploys toxins into target cells. Surprisingly, displacement was independent of T6SS-mediated killing of Aeromonas, driven instead by T6SS-induced enhancement of zebrafish intestinal movements that led to expulsion of the resident commensal by the host. Deleting an actin crosslinking domain from the T6SS apparatus returned intestinal motility to normal and thwarted expulsion, without weakening V. cholerae's ability to kill Aeromonas in vitro. Our finding that bacteria can manipulate host physiology to influence inter-microbial competition has implications for both pathogenesis and microbiome engineering.   

Universality of clone dynamics during tissue development

Lineage tracing studies based on transgenic animal models have led to advances in our understanding of cellular identity, hierarchy and function. They provide insights into the development, maintenance and regeneration of tissues, and factors leading to dysregulation in diseased states. However, large-scale cell rearrangements, particularly in growing tissues may render the retrospective analysis of lineages highly problematic. Drawing on studies of heart development, we show how such effects may lead to the emergence of universal scaling distributions. By mapping the problem of clonal evolution onto the theory of aerosols, we elucidate the origin and range of possible scaling behaviors. In generalizing our studies to other tissue types and contexts, we show how the identification of universal scaling dependences allows biological information on cell fate behavior to be distilled.