Publications

The F-BAR protein Cdc15 is essential for cytokinesis in Schizosaccharomyces pombe and plays a key role in attaching the cytokinetic ring (CR) to the plasma membrane (PM). Cdc15’s abilities to bind to the membrane and oligomerize via its F-BAR domain are inhibited by phosphorylation of its intrinsically disordered region (IDR). Multiple cell polarity kinases regulate Cdc15 IDR phosphostate, and of these the DYRK kinase Pom1 phosphorylation sites on Cdc15 have been shown in vivo to prevent CR formation at cell tips. Here, we compared the ability of Pom1 to control Cdc15 phosphostate and cortical localization to that of other Cdc15 kinases: Kin1, Pck1, and Shk1. We identified distinct but overlapping cohorts of Cdc15 phosphorylation sites targeted by each kinase, and the number of sites correlated with each kinases’ abilities to influence Cdc15 PM localization. Coarse-grained simulations predicted that cumulative IDR phosphorylation moves the IDRs of a dimer apart and toward the F-BAR tips. Further, simulations indicated that the overall negative charge of phosphorylation masks positively charged amino acids necessary for F-BAR oligomerization and membrane interaction. Finally, simulations suggested that dephosphorylated Cdc15 undergoes phase separation driven by IDR interactions. Indeed, dephosphorylated but not phosphorylated Cdc15 undergoes liquid–liquid phase separation to form droplets in vitro that recruit Cdc15 binding partners. In cells, Cdc15 phosphomutants also formed PM-bound condensates that recruit other CR components. Together, we propose that a threshold of Cdc15 phosphorylation by assorted kinases prevents Cdc15 condensation on the PM and antagonizes CR assembly.

Segmentation of the Drosophila embryo is initiated by localized maternal signals. In this context, anteriorly localized Bicoid activates the gap genes in the anterior half of the embryo while posteriorly localized Nanos represses the translation of maternal hunchback mRNA to pattern the posterior half. The non-segmented termini are patterned by the localized activation of mitogen-activated protein kinase. Yet, the spatial extent of the terminal patterning system in regulating gap genes beyond poles remains unknown. We investigated the patterning potential of the terminal system using mutagenized embryos that lack both the anterior and the posterior maternal signaling systems. Using a combination of quantitative imaging and mathematical modeling, we analyzed the spatial patterns of gap genes in the early Drosophila embryo. We found that this mutant embryo develops symmetric cuticle patterns along the anteroposterior axis with two segments on each side. Notably, the terminal system can affect the expression of Krüppel in the torso region. Our mathematical model recapitulates the experimental data and reveals the potential bistability in the terminal patterning system. Collectively, our study suggests that the terminal system can act as a long-range inductive signal and establish multiple gene expression boundaries along the anteroposterior axis of the developing embryo.

The previously reported Q is a thermoresponsive coiled-coil protein capable of higher-order supramolecular assembly into fibers and hydrogels with upper critical solution temperature (UCST) behavior. Here, we introduce a new coiled-coil protein that is redesigned to disfavor lateral growth of its fibers and thus achieve a higher crosslinking density within the formed hydrogel. We also introduce a favorable hydrophobic mutation to the pore of the coiled-coil domain for increased thermostability of the protein. We note that an increase in storage modulus of the hydrogel and crosslinking density is coupled with a decrease in fiber diameter. We further fully characterize our α-helical coiled-coil (Q2) hydrogel for its structure, nano-assembly, and rheology relative to our previous single domain protein, Q, over the time of its gelation demonstrating the nature of our hydrogel self-assembly system. In this vein, we also characterize the ability of Q2 to encapsulate the small hydrophobic small molecule, curcumin, and its impact on the mechanical properties of Q2. The design parameters here not only show the importance of electrostatic potential in self-assembly but also provide a step towards predictable design of electrostatic protein interactions.

Drosophila oogenesis is a powerful and tractable model for studies of cell and developmental biology due to the multitude of well-characterized events in both germline and somatic cells, the ease of genetic manipulation in fruit flies, and the large number of egg chambers produced by each fly. Recent improvements in live imaging and ex vivo culturing protocols have enabled researchers to conduct more detailed, longer-term studies of egg chamber development, enabling insights into fundamental biological processes. Here, we present a protocol for dissection, culturing, and imaging of late-stage egg chambers to study intercellular and directional cytoplasmic flow during “nurse cell dumping.” This critical developmental process towards the latter stages of oogenesis (stages 10b/11) results in rapid growth of the oocyte and shrinkage of the nurse cells and is accompanied by dynamic changes in cell shape.

Fluorescent protein biomaterials have important applications such as bioimaging in pharmacological studies. Self-assembly of proteins, especially into fibrils, is known to produce fluorescence in the blue band. Capable of self-assembly into nanofibers, we have shown we can modulate its aggregation into mesofibers by encapsulation of a small hydrophobic molecule. Conversely, azobenzenes are hydrophobic small molecules that are virtually non-fluorescent in solution due to their highly efficient photoisomerization. However, they demonstrate fluorogenic properties upon confinement in nanoscale assemblies by reducing the non-radiative photoisomerization. Here, we report the fluorescence of a hybrid protein-small molecule system in which azobenzene is confined in our protein assembly leading to fiber thickening and increased fluorescence. We show our engineered protein Q encapsulates AzoCholine, bearing a photoswitchable azobenzene moiety, in the hydrophobic pore to produce fluorescent mesofibers. This study further investigates the photocontrol of protein conformation as well as fluorescence of an azobenze-containing biomaterial.

The fluid–structure interactions between flexible fibres and viscous flows play an essential role in various biological phenomena, medical problems and industrial processes. Of particular interest is the case of particles transported freely in time-dependent flows. This work elucidates the dynamics and morphologies of actin filaments under oscillatory shear flows by combining microfluidic experiments, numerical simulations and theoretical modelling. Our work reveals that, in contrast to steady shear flows, in which small orientational fluctuations from a flow-aligned state initiate tumbling and deformations, the periodic flow reversal allows the filament to explore many different configurations at the beginning of each cycle. Investigation of filament motion during half time periods of oscillation highlights the critical role of the initial filament orientation on the emergent dynamics. This strong coupling between orientation and deformation results in new deformation regimes and novel higher-order buckling modes absent in steady shear flows. The primary outcome of our analysis is the possibility of suppression of buckling instabilities for certain combinations of the oscillation frequency and initial filament orientation, even in very strong flows. We explain this unusual behaviour through a weakly nonlinear Landau theory of buckling, in which we treat the filaments as inextensible Brownian Euler–Bernoulli rods whose hydrodynamics is described by local slender-body theory.

The nucleation of actin filament branches by the Arp2/3 complex involves activation through nucleation promotion factors (NPFs), recruitment of actin monomers, and binding of the complex to the side of actin filaments. Because of the large system size and processes that involve flexible regions and diffuse components, simulations of branch formation using all-atom molecular dynamics are challenging. We applied a coarse-grained model that retains amino-acid level information and allows molecular dynamics simulations in implicit solvent, with globular domains represented as rigid bodies and flexible regions allowed to fluctuate. We used recent electron microscopy structures of the inactive Arp2/3 complex bound to NPF domains and to mother actin filament for the activated Arp2/3 complex. We studied interactions of Arp2/3 complex with the activating VCA domain of the NPF Wiskott-Aldrich syndrome protein, actin monomers, and actin filament. We found stable configurations with one or two actin monomers bound along the branch filament direction and with CA domain of VCA associated to the strong and weak binding sites of the Arp2/3 complex, supporting prior structural studies and validating our approach. We reproduced delivery of actin monomers and CA to the Arp2/3 complex under different conditions, providing insight into mechanisms proposed in previous studies. Simulations of active Arp2/3 complex bound to a mother actin filament indicate the contribution of each subunit to the binding. Addition of the C-terminal tail of Arp2/3 complex subunit ArpC2, which is missing in the cryo-EM structure, increased binding affinity, indicating a possible stabilizing role of this tail.

Recent experiments have shown that the nematode {\it T. aceti} can assemble into collectively undulating groups at the edge of fluid drops. This coordinated state consists of metachronal waves and drives fluid circulation inside the drop. We find that the circulation velocity is about 2 mm/s and nearly half the speed of the metachronal wave. We develop a quasi two-dimensional hydrodynamics model using the Stokes flow approximation. The periodic motion of the nematodes constitute our moving boundary condition that drives the flow. Our model suggests that large amplitude excursions of the nematodes tails produce the fluid circulation. We discuss the constraints on containers that would enhance fluid motion, which could be used in the future design of on demand flow generating systems.

The rupture potential of an atherosclerotic plaque is dependent on both the plaque's composition and the shear stresses it encounters from blood flow. Because plaques move and deform throughout the cardiac cycle, resulting in changes to plaque position and shape as well as to the encountered shear stresses, concurrent imaging of both risk factors over time is required to accurately predict plaque vulnerability. To evaluate the potential to achieve as much, multi-angle plane wave (PW) ARFI and least-squares vector Doppler data were acquired in a calibrated flow phantom with channels of 4–8 mm diameters and flow rates of 100–600 ml/min. The wall shear stress (WSS) was measured to within 15% of the ground-truth analytical solutions. The same methods were then implemented in an excised human cadaveric carotid with a x% stenotic plaque. ARFI VoA detected plaque regions of calcium and intraplaque hemorrhage that were validated by spatially-matched histology. Concurrent vector Doppler yielded a peak WSS of 5.2 Pa on the plaque shoulder, which was consistent with the 6.4 Pa WSS predicted by an immersed interface fluid-solid interation (FSI) model developed using the specific geometry of the examined cadaveric carotid. Overall our results demonstrate the feasibility of concurrent imaging of carotid plaque composition by ARFI VoA, vector flow, and WSS to better assess stroke risk.