Defining the proximal interaction networks of Arf GTPases reveals a mechanism for the regulation of PLD1 and PI4KB.

The Arf GTPase family is involved in a wide range of cellular regulation including membrane trafficking and organelle-structure assembly. Here, we have generated a proximity interaction network for the Arf family using the miniTurboID approach combined with TMT-based quantitative mass spectrometry. Our interactome confirmed known interactions and identified many novel interactors that provide leads for defining Arf pathway cell biological functions. We explored the unexpected finding that phospholipase D1 (PLD1) preferentially interacts with two closely related but poorly studied Arf family GTPases, ARL11 and ARL14, showing that PLD1 is activated by ARL11/14 and may recruit these GTPases to membrane vesicles, and that PLD1 and ARL11 collaborate to promote macrophage phagocytosis. Moreover, ARL5A and ARL5B were found to interact with and recruit phosphatidylinositol 4-kinase beta (PI4KB) at trans-Golgi, thus promoting PI4KB's function in PI4P synthesis and protein secretion.

Hippo pathway regulation by phosphatidylinositol transfer protein and phosphoinositides.

The Hippo pathway plays a key role in development, organ size control and tissue homeostasis, and its dysregulation contributes to cancer. The LATS tumor suppressor kinases phosphorylate and inhibit the YAP/TAZ transcriptional co-activators to suppress gene expression and cell growth. Through a screen of marine natural products, we identified microcolin B (MCB) as a Hippo activator that preferentially kills YAP-dependent cancer cells. Structure-activity optimization yielded more potent MCB analogs, which led to the identification of phosphatidylinositol transfer proteins α and ß (PITPα/ß) as the direct molecular targets. We established a critical role of PITPα/ß in regulating LATS and YAP. Moreover, we showed that PITPα/ß influence the Hippo pathway via plasma membrane phosphatidylinositol-4-phosphate. This study uncovers a previously unrecognized role of PITPα/ß in Hippo pathway regulation and as potential cancer therapeutic targets.

Strong triaxial coupling and anomalous Poisson effect in collagen networks.

While cells within tissues generate and sense 3D states of strain, the current understanding of the mechanics of fibrous extracellular matrices (ECMs) stems mainly from uniaxial, biaxial, and shear tests. Here, we demonstrate that the multiaxial deformations of fiber networks in 3D cannot be inferred solely based on these tests. The interdependence of the three principal strains gives rise to anomalous ratios of biaxial to uniaxial stiffness between 8 and 9 and apparent Poisson's ratios larger than 1. These observations are explained using a microstructural network model and a coarse-grained constitutive framework that predicts the network Poisson effect and stress-strain responses in uniaxial, biaxial, and triaxial modes of deformation as a function of the microstructural properties of the network, including fiber mechanics and pore size of the network. Using this theoretical approach, we found that accounting for the Poisson effect leads to a 100-fold increase in the perceived elastic stiffness of thin collagen samples in extension tests, reconciling the seemingly disparate measurements of the stiffness of collagen networks using different methods. We applied our framework to study the formation of fiber tracts induced by cellular forces. In vitro experiments with low-density networks showed that the anomalous Poisson effect facilitates higher densification of fibrous tracts, associated with the invasion of cancerous acinar cells. The approach developed here can be used to model the evolving mechanics of ECM during cancer invasion and fibrosis.

A fluorogenic array for temporally unlimited single-molecule tracking.

We describe three optical tags, ArrayG, ArrayD and ArrayG/N, for intracellular tracking of single molecules over milliseconds to hours. ArrayG is a fluorogenic tag composed of a green fluorescent protein-nanobody array and monomeric wild-type green fluorescent protein binders that are initially dim but brighten ~26-fold on binding with the array. By balancing the rates of binder production, photobleaching and stochastic binder exchange, we achieve temporally unlimited tracking of single molecules. High-speed tracking of ArrayG-tagged kinesins and integrins for thousands of frames reveals novel dynamical features. Tracking of single histones at 0.5 Hz for >1 hour with the import competent ArrayG/N tag shows that chromosomal loci behave as Rouse polymers with visco-elastic memory and exhibit a non-Gaussian displacement distribution. ArrayD, based on a dihydrofolate reductase nanobody array and dihydrofolate reductase-fluorophore binder, enables dual-color imaging. The arrays combine brightness, fluorogenicity, fluorescence replenishment and extended fluorophore choice, opening new avenues for tracking single molecules in living cells.

NuSeT: A deep learning tool for reliably separating and analyzing crowded cells.

Segmenting cell nuclei within microscopy images is a ubiquitous task in biological research and clinical applications. Unfortunately, segmenting low-contrast overlapping objects that may be tightly packed is a major bottleneck in standard deep learning-based models. We report a Nuclear Segmentation Tool (NuSeT) based on deep learning that accurately segments nuclei across multiple types of fluorescence imaging data. Using a hybrid network consisting of U-Net and Region Proposal Networks (RPN), followed by a watershed step, we have achieved superior performance in detecting and delineating nuclear boundaries in 2D and 3D images of varying complexities. By using foreground normalization and additional training on synthetic images containing non-cellular artifacts, NuSeT improves nuclear detection and reduces false positives. NuSeT addresses common challenges in nuclear segmentation such as variability in nuclear signal and shape, limited training sample size, and sample preparation artifacts. Compared to other segmentation models, NuSeT consistently fares better in generating accurate segmentation masks and assigning boundaries for touching nuclei.

Mechanisms of Plastic Deformation in Collagen Networks Induced by Cellular Forces.

Contractile cells can reorganize fibrous extracellular matrices and form dense tracts of fibers between neighboring cells. These tracts guide the development of tubular tissue structures and provide paths for the invasion of cancer cells. Here, we studied the mechanisms of the mechanical plasticity of collagen tracts formed by contractile premalignant acinar cells and fibroblasts. Using fluorescence microscopy and second harmonic generation, we quantified the collagen densification, fiber alignment, and strains that remain within the tracts after cellular forces are abolished. We explained these observations using a theoretical fiber network model that accounts for the stretch-dependent formation of weak cross-links between nearby fibers. We tested the predictions of our model using shear rheology experiments. Both our model and rheological experiments demonstrated that increasing collagen concentration leads to substantial increases in plasticity. We also considered the effect of permanent elongation of fibers on network plasticity and derived a phase diagram that classifies the dominant mechanisms of plasticity based on the rate and magnitude of deformation and the mechanical properties of individual fibers. Plasticity is caused by the formation of new cross-links if moderate strains are applied at small rates or due to permanent fiber elongation if large strains are applied over short periods. Finally, we developed a coarse-grained model for plastic deformation of collagen networks that can be employed to simulate multicellular interactions in processes such as morphogenesis, cancer invasion, and fibrosis.

Insights into recent findings and clinical application of YAP and TAZ in cancer.

Decades of research have mapped out the basic mechanics of the Hippo pathway. The paralogues Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), as the central transcription control module of the Hippo pathway, have long been implicated in the progression of various human cancers. The current literature regarding oncogenic YAP and TAZ activities consists mostly of context-specific mechanisms and treatments of human cancers. Furthermore, a growing number of studies demonstrate tumour-suppressor functions of YAP and TAZ. In this Review we aim to synthesize an integrated perspective of the many disparate findings regarding YAP and TAZ in cancer. We then conclude with the various strategies for targeting and treating YAP- and TAZ-dependent cancers.

Numerical validation of IFT in the analysis of protein-surfactant complexes with SAXS and SANS.

The use of the indirect Fourier transform methods for evaluating structural parameters directly in real space with small-angle scattering measurements is validated for the analysis of protein-surfactant complexes. An efficient Monte Carlo approach rapidly generates in silico structures based on a realistic pearl-necklace model for denatured proteins decorated with surfactant micelles. Corresponding scattering profiles are calculated and averaged over a large number of possible configurations for each structure. IFT algorithms are then used to calculate the corresponding pair-distance distribution function, and structural information is extracted directly without model fitting. The extracted parameters are compared and correlated with the known structure of the simulated complexes to assess the quality of the information that can be reliably obtained from these systems. The average extension, nearest-neighbor micelle distance, and average number of associated micelles are all accurately extracted through IFT calculations. Improved and simple approaches to reliably extract the average extension of the complex and the total number of associated micelles are presented.

Concerted localization-resets precede YAP-dependent transcription.

Yes-associated protein 1 (YAP) is a transcriptional regulator with critical roles in mechanotransduction, organ size control, and regeneration. Here, using advanced tools for real-time visualization of native YAP and target gene transcription dynamics, we show that a cycle of fast exodus of nuclear YAP to the cytoplasm followed by fast reentry to the nucleus ("localization-resets") activates YAP target genes. These "resets" are induced by calcium signaling, modulation of actomyosin contractility, or mitosis. Using nascent-transcription reporter knock-ins of YAP target genes, we show a strict association between these resets and downstream transcription. Oncogenically-transformed cell lines lack localization-resets and instead show dramatically elevated rates of nucleocytoplasmic shuttling of YAP, suggesting an escape from compartmentalization-based control. The single-cell localization and transcription traces suggest that YAP activity is not a simple linear function of nuclear enrichment and point to a model of transcriptional activation based on nucleocytoplasmic exchange properties of YAP.