Cytoplasmic dynein is a microtubule-based motor required for intracellular transport and cell division. Its movement involves coupling cycles of track binding and release with cycles of force-generating nucleotide hydrolysis. How this is accomplished given the ~25 nm separating dynein’s track- and nucleotide-binding sites is not understood. Here, we present a sub-nanometer-resolution structure of dynein’s microtubule-binding domain bound to microtubules by cryo-electron microscopy that was used to generate a pseudo-atomic model of the complex with molecular dynamics. We identified large rearrangements triggered by track binding and specific interactions, confirmed by mutagenesis and single molecule motility assays, which tune dynein’s affinity for microtubules. Our results provide a molecular model for how dynein’s binding to microtubules is communicated to the rest of the motor.

Overexpressed tumor associated antigens (e.g., HER2 and epidermal growth factor receptor) are attractive targets for therapeutic T cells, but toxic “off-tumor” cross-reaction with normal tissues expressing low levels of target antigen can occur with Chimeric Antigen Receptor (CAR) T cells. Inspired by natural ultrasensitive response circuits, we engineered a two-step positive feedback circuit that allows T cells to discriminate targets based on a sigmoidal antigen density threshold. In this circuit, a low affinity synthetic Notch receptor for HER2 controls the expression of a high affinity CAR for HER2. Increasing HER2 density thus has cooperative effects on T cells—it both increases CAR expression and activation—leading to a sigmoidal response. T cells with this circuit show sharp discrimination between target cells expressing normal amounts of HER2 and cancer cells expressing 100-fold more HER2, both in vitro and in vivo.

Cells control organelle size with great precision and accuracy to maintain optimal physiology, but the mechanisms by which they do so are largely unknown. Cilia and flagella are simple organelles in which a single measurement, length, can represent size. Maintenance of flagellar length requires an active transport process known as intraflagellar transport, and previous measurements suggest that a length-dependent feedback regulates intraflagellar transport. But the question remains: how is a length-dependent signal produced to regulate intraflagellar transport appropriately? Several conceptual models have been suggested, but testing these models quantitatively requires that they be cast in mathematical form. Here, we derive a set of mathematical models that represent the main broad classes of hypothetical size-control mechanisms currently under consideration. We use these models to predict the relation between length and intraflagellar transport, and then compare the predicted relations for each model with experimental data. We find that three models-an initial bolus formation model, an ion current model, and a diffusion-based model-show particularly good agreement with available experimental data. The initial bolus and ion current models give mathematically equivalent predictions for length control, but fluorescence recovery after photobleaching experiments rule out the initial bolus model, suggesting that either the ion current model or a diffusion-based model is more likely correct. The general biophysical principles of the ion current and diffusion-based models presented here to measure cilia and flagellar length can be generalized to measure any membrane-bound organelle volume, such as the nucleus and endoplasmic reticulum.

Biomaterials can improve the safety and presentation of therapeutic agents for effective immunotherapy, and a high level of control over surface functionalization is essential for immune cell modulation. Here, we developed biocompatible immune cell engaging particles (ICEp) that use synthetic short DNA as scaffolds for efficient and tunable protein loading. To improve the safety of chimeric antigen receptor (CAR) T cell therapies, micron-sized ICEp were injected intratumorally to present a priming signal for systemically administered AND-gate CAR-T cells. Locally retained ICEp presenting a high density of priming antigens activated CAR-T cells, driving local tumor clearance while sparing uninjected tumors in immunodeficient mice. The ratiometric control of costimulatory ligands (anti-CD3 and anti-CD28 antibodies) and the surface presentation of a cytokine (IL-2) on ICEp were shown to significantly impact human primary T cell activation phenotypes. This modular and versatile biomaterial functionalization platform can provide new opportunities for immunotherapies.

Overexpressed tumor associated antigens (e.g. HER2 and EGFR) are attractive targets for therapeutic T cells, but toxic cross-reaction with normal tissues expressing low antigen levels has been observed with Chimeric Antigen Receptor (CAR) T cells targeting such antigens. Inspired by natural ultrasensitive response circuits, we engineer a two-step positive feedback circuit that allows T cells to discriminate targets based on a sigmoidal antigen density threshold. In this circuit, a low affinity SynNotch receptor for HER2 controls the expression of a high affinity CAR for HER2. Increasing HER2 density thus has cooperative effects on T cells ╌ it both increases CAR expression and activation ╌ leading to a sigmoidal response. T Cells with this circuit show sharp discrimination between target cells expressing normal and disease levels of HER2, both in vitro and in vivo.One Sentence SummaryA two-step positive feedback circuit generates engineered T cells capable of killing target cells with an ultrasensitive antigen density threshold.