pHusion - a robust and versatile toolset for automated detection and analysis of exocytosis.

Exocytosis is a fundamental process used by eukaryotes to regulate the composition of the plasma membrane and facilitate cell-cell communication. To investigate exocytosis in neuronal morphogenesis, previously we developed computational tools with a graphical user interface to enable the automatic detection and analysis of exocytic events from fluorescence timelapse images. Although these tools were useful, we found the code was brittle and not easily adapted to different experimental conditions. Here, we developed and validated a robust and versatile toolkit, named pHusion, for the analysis of exocytosis, written in ImageTank, a graphical programming language that combines image visualization and numerical methods. We tested pHusion using a variety of imaging modalities and pH-sensitive fluorophores, diverse cell types and various exocytic markers, to generate a flexible and intuitive package. Using this system, we show that VAMP3-mediated exocytosis occurs 30-times more frequently in melanoma cells compared with primary oligodendrocytes, that VAMP2-mediated fusion events in mature rat hippocampal neurons are longer lasting than those in immature murine cortical neurons, and that exocytic events are clustered in space yet random in time in developing cortical neurons.

TRIM9 controls growth cone responses to netrin through DCC and UNC5C.

The guidance cue netrin-1 promotes both growth cone attraction and growth cone repulsion. How netrin-1 elicits these diverse axonal responses, beyond engaging the attractive receptor DCC and repulsive receptors of the UNC5 family, remains elusive. Here we demonstrate that murine netrin-1 induces biphasic axonal responses in cortical neurons: attraction at lower concentrations and repulsion at higher concentrations using both a microfluidic-based netrin-1 gradient and bath application of netrin-1. TRIM9 is a brain-enriched E3 ubiquitin ligase previously shown to bind and cluster the attractive receptor DCC at the plasma membrane and regulate netrin-dependent attractive responses. However, whether TRIM9 also regulated repulsive responses to netrin-1 remained to be seen. In this study, we show that TRIM9 localizes and interacts with both the attractive netrin receptor DCC and the repulsive netrin receptor, UNC5C, and that deletion of murine Trim9 alters both attractive and repulsive responses to murine netrin-1. TRIM9 was required for netrin-1-dependent changes in surface levels of DCC and total levels of UNC5C in the growth cone during morphogenesis. We demonstrate that DCC at the membrane regulates growth cone area and show that TRIM9 negatively regulates FAK activity in the absence of netrin-1. We investigate membrane dynamics of the UNC5C receptor using pH-mScarlet fused to the extracellular domain of UNC5C. Minutes after netrin addition, levels of UNC5C at the plasma membrane drop in a TRIM9-independent fashion, however TRIM9 regulated the mobility of UNC5C in the plasma membrane in the absence of netrin-1. Together this work demonstrates that TRIM9 interacts with and regulates both DCC and UNC5C during attractive and repulsive axonal responses to netrin-1.

4D intravital imaging studies identify platelets as the predominant cellular procoagulant surface in a mouse model of hemostasis.

Interplay between platelets, coagulation/fibrinolytic factors, and endothelial cells (ECs) is necessary for effective hemostatic plug formation. This study describes a novel four-dimensional (4D) imaging platform to visualize and quantify hemostatic plug components with high spatiotemporal resolution. Fibrin accumulation following laser-induced endothelial ablation was observed at the EC-platelet plug interface, controlled by the antagonistic balance between fibrin generation and breakdown. Phosphatidylserine (PS) was first detected in close physical proximity to the fibrin ring, followed by exposure across the endothelium. Impaired PS exposure in cyclophilinD -/- mice resulted in a significant reduction in fibrin accumulation. Adoptive transfer and inhibitor studies demonstrated a key role for platelets, but not ECs, in fibrin generation during hemostatic plug formation. Inhibition of fibrinolysis with tranexamic acid (TXA) led to increased fibrin accumulation in WT mice, but not in cyclophilinD -/- mice or WT mice treated with antiplatelet drugs. These studies implicate platelets as the functionally dominant procoagulant surface during hemostatic plug formation. In addition, they suggest that impaired fibrin formation due to reduced platelet procoagulant activity is not reversed by TXA treatment.

pHusion: A robust and versatile toolset for automated detection and analysis of exocytosis.

Exocytosis is a fundamental process used by eukaryotic cells to regulate the composition of the plasma membrane and facilitate cell-cell communication. To investigate the role exocytosis plays in neuronal morphogenesis, previously we developed computational tools with a graphical user interface (GUI) to enable the automatic detection and analysis of exocytic events (ADAE GUI) from fluorescence timelapse images. Though these tools have proven useful, we found that the code was brittle and not easily adapted to different experimental conditions. Here, we have developed and validated a robust and versatile toolkit, named pHusion, for the analysis of exocytosis written in ImageTank, a graphical programming language that combines image visualization and numerical methods. We tested this method using a variety of imaging modalities and pH-sensitive fluorophores, diverse cell types, and various exocytic markers to generate a flexible and intuitive package. Using pHusion, we show that VAMP3-mediated exocytosis occurs 30-times more frequently in melanoma cells compared with primary oligodendrocytes, that VAMP2-mediated fusion events in mature rat hippocampal neurons are longer lasting than those in immature murine cortical neurons, and that exocytic events are clustered in space yet random in time in developing cortical neurons. Summary Statement: Exocytosis is an essential process by which cells change shape, alter membrane composition, and communicate with other cells. Though all eukaryotic cells carry out exocytosis, the regulation of vesicle fusion, the cargo of vesicles, and the role exocytosis plays in cell fate differ greatly across cell types. Here, we developed a flexible and robust set of tools to enable automatic identification and analysis of exocytic events across a wide range of cell types, vesicle types, and imaging conditions.

A multi-functional microfluidic device compatible with widefield and light sheet microscopy.

We present a microfluidic device compatible with high resolution light sheet and super-resolution microscopy. The device is a 150 µm thick chamber with a transparent fluorinated ethylene propylene (FEP) cover that has a similar refractive index (1.34) to water (1.33), making it compatible with top-down imaging used in light sheet microscopy. We provide a detailed fabrication protocol and characterize the optical performance of the device. We demonstrate that the device supports long-term imaging of cell growth and differentiation as well as the rapid addition and removal of reagents while simultaneously maintaining sterile culture conditions by physically isolating the sample from the dipping lenses used for imaging. Finally, we demonstrate that the device can be used for super-resolution imaging using lattice light sheet structured illumination microscopy (LLS-SIM) and DNA PAINT. We anticipate that FEP-based microfluidics, as shown here, will be broadly useful to researchers using light sheet microscopy due to the ability to switch reagents, image weakly adherent cells, maintain sterility, and physically isolate the specimen from the optics of the instruments.

Software for lattice light-sheet imaging of FRET biosensors, illustrated with a new Rap1 biosensor.

Lattice light-sheet microscopy (LLSM) is valuable for its combination of reduced photobleaching and outstanding spatiotemporal resolution in 3D. Using LLSM to image biosensors in living cells could provide unprecedented visualization of rapid, localized changes in protein conformation or posttranslational modification. However, computational manipulations required for biosensor imaging with LLSM are challenging for many software packages. The calculations require processing large amounts of data even for simple changes such as reorientation of cell renderings or testing the effects of user-selectable settings, and lattice imaging poses unique challenges in thresholding and ratio imaging. We describe here a new software package, named ImageTank, that is specifically designed for practical imaging of biosensors using LLSM. To demonstrate its capabilities, we use a new biosensor to study the rapid 3D dynamics of the small GTPase Rap1 in vesicles and cell protrusions.

Functional redundancy between RAP1 isoforms in murine platelet production and function.

RAP GTPases, important regulators of cellular adhesion, are abundant signaling molecules in the platelet/megakaryocytic lineage. However, mice lacking the predominant isoform, RAP1B, display a partial platelet integrin activation defect and have a normal platelet count, suggesting the existence of a RAP1-independent pathway to integrin activation in platelets and a negligible role for RAP GTPases in megakaryocyte biology. To determine the importance of individual RAP isoforms on platelet production and on platelet activation at sites of mechanical injury or vascular leakage, we generated mice with megakaryocyte-specific deletion (mKO) of Rap1a and/or Rap1b Interestingly, Rap1a/b-mKO mice displayed a marked macrothrombocytopenia due to impaired proplatelet formation by megakaryocytes. In platelets, RAP isoforms had redundant and isoform-specific functions. Deletion of RAP1B, but not RAP1A, significantly reduced α-granule secretion and activation of the cytoskeleton regulator RAC1. Both isoforms significantly contributed to thromboxane A2 generation and the inside-out activation of platelet integrins. Combined deficiency of RAP1A and RAP1B markedly impaired platelet aggregation, spreading, and clot retraction. Consistently, thrombus formation in physiological flow conditions was abolished in Rap1a/b-mKO, but not Rap1a-mKO or Rap1b-mKO, platelets. Rap1a/b-mKO mice were strongly protected from experimental thrombosis and exhibited a severe defect in hemostasis after mechanical injury. Surprisingly, Rap1a/b-mKO platelets were indistinguishable from controls in their ability to prevent blood-lymphatic mixing during development and hemorrhage at sites of inflammation. In summary, our studies demonstrate an essential role for RAP1 signaling in platelet integrin activation and a critical role in platelet production. Although important for hemostatic/thrombotic plug formation, platelet RAP1 signaling is dispensable for vascular integrity during development and inflammation.

Analyzing and engineering cell signaling modules with synthetic biology.

Signaling pathways lie at the heart of cellular responses to environmental cues. The ability to reconstruct specific signaling modules ex vivo allows us to study their inherent properties in an isolated environment, which in turn enables us to elucidate fundamental design principles for such motifs. This synthetic biology approach for analyzing natural, well-defined signaling modules will help to bridge the gap between studies on isolated biochemical reactions-which can provide great mechanistic detail but do not capture the complexity of endogenous signaling pathways-and those on entire networks of protein interactions-which offer a systems-level view of signal transduction but obscure the mechanisms that underlie signal transmission and processing. Additionally, minimal signaling modules can be tractably engineered to predictably alter cellular responses, opening up possibilities for creating biotechnologically and biomedically useful cellular devices.

Tunable signal processing in synthetic MAP kinase cascades.

The flexibility of MAPK cascade responses enables regulation of a vast array of cell fate decisions, but elucidating the mechanisms underlying this plasticity is difficult in endogenous signaling networks. We constructed insulated mammalian MAPK cascades in yeast to explore how intrinsic and extrinsic perturbations affect the flexibility of these synthetic signaling modules. Contrary to biphasic dependence on scaffold concentration, we observe monotonic decreases in signal strength as scaffold concentration increases. We find that augmenting the concentration of sequential kinases can enhance ultrasensitivity and lower the activation threshold. Further, integrating negative regulation and concentration variation can decouple ultrasensitivity and threshold from the strength of the response. Computational analyses show that cascading can generate ultrasensitivity and that natural cascades with different kinase concentrations are innately biased toward their distinct activation profiles. This work demonstrates that tunable signal processing is inherent to minimal MAPK modules and elucidates principles for rational design of synthetic signaling systems.