Biology subjects

Welf, E. S.

Publications and source records attributed to Welf, E. S..

11 recordsLinked to original sources

bioRxiv2021

Blebs Promote Cell Survival by Assembling Oncogenic Signaling Hubs

Most human cells require anchorage for survival. Cell-substrate adhesion activates diverse signaling pathways, without which cells undergo anoikis - a form of programmed cell death1. Acquisition of anoikis resistance is a pivotal step in cancer disease progression, as metastasizing cancer cells often lose firm attachment to surrounding tissue2-5. In these poorly attached states, cells adopt rounded morphologies and form small hemispherical plasma membrane protrusions called blebs6-13. Bleb function has been thoroughly investigated in the context of amoeboid migration but is far less examined in other scenarios14-19. Here we show by quantitative subcellular 3D imaging and manipulation of cell morphological states that blebbing triggers the formation of plasma membrane-proximal signaling hubs that confer anoikis resistance. Specifically, we discovered in melanoma cells that blebbing generates plasma membrane contours, which recruit curvature-sensing septin proteins as scaffolds for constitutively active mutant NRAS and effectors. These signaling hubs activate ERK and PI3K - canonical promoters of pro-survival pathways. Inhibition of blebs or septins has little effect on the survival of well-adhered cells, but in detached cells causes NRAS mislocalization, reduced MAPK and PI3K activity, and ultimately, death. This unveils an unanticipated morphological requirement for mutant NRAS to operate as an effective oncoprotein. Moreover, we find that though some BRAF mutant melanoma do not rely on this survival pathway in a basal state, BRAF/MEK inhibition strongly sensitizes them to both bleb and septin inhibition. Importantly, we demonstrate that fibroblasts engineered to sustain blebbing acquire the same anoikis resistance as cancer cells even without harboring oncogenic mutations. These data define a role for blebs as potent signaling organelles capable of integrating myriad cellular information flows into concerted cellular responses, in this case granting robust anoikis resistance.

cell biology↗

bioRxiv2021

Cancer drug discovery as a low rank tensor completion problem

The heterogeneity of cancer necessitates developing a multitude of targeted therapies. We propose the view that cancer drug discovery is a low rank tensor completion problem. We implement this vision by using heterogeneous public data to construct a tensor of drug-target-disease associations. We show the validity of this approach computationally by simulations, and experimentally by testing drug candidates. Specifically, we show that a novel drug candidate, SU11652, controls melanoma tumor growth, including BRAFWT melanoma. Independently, we show that another molecule, TC-E 5008, controls tumor proliferation on ex vivo ER+ human breast cancer. Most importantly, we identify these chemicals with only a few computationally selected experiments as opposed to brute-force screens. The efficiency of our approach enables use of ex vivo human tumor assays as a primary screening tool. We provide a web server, the Cancer Vulnerability Explorer (accessible at https://cavu.biohpc.swmed.edu), to facilitate the use of our methodology.

cancer biology↗

bioRxiv2020

u-track 3D: measuring and interrogating intracellular dynamics in three dimensions.

Particle tracking is a ubiquitous task in the study of dynamic molecular and cellular processes through microscopy. Light-sheet microscopy has opened a path to acquiring complete cell volumes for investigation in 3-dimensions (3D). However, hypothesis formulation and quantitative analysis have remained difficult due to fundamental challenges in the visualization and the verification of large and dense sets of 3D particle trajectories. Here we describe u-track 3D, a software package that addresses these two challenges. Building on the established framework of particle association in space and time implemented for 2D time-lapse sequences, we first report a complete and versatile pipeline for particle tracking in 3D. We then present the concept of dynamic region of interest (dynROI), which allows an experimenter to interact with dynamic 3D processes in 2D views amenable to visual inspection. Third, we present an estimator of trackability which defines a score for every trajectory, thereby overcoming the challenges of trajectory validation by visual inspection. With these combined strategies, u-track 3D provides a framework for the unbiased study of molecular processes in complex volumetric sequences.

cell biology↗

bioRxiv2020

Worrying drives cell migration in mechanically unrestrained environments

Migratory cells often encounter crowded microenvironments through which they must find or make a path. Amoeboid cells are thought to find a path by deforming their bodies to squeeze through tight spaces. Yet many amoeboid cells seem to maintain a near spherical morphology as they move. To examine this unexplored mechanism of migration, we visualized amoeboid melanoma cells in dense environments and found that they carve a path via bleb-driven mechanical degradation of extracellular matrix components without proteolytic degradation. Interactions between adhesions and collagen at the cell front induce a signaling cascade that promotes bleb enlargement via branched actin polymerization. Large blebs abrade collagen, creating feedback between extracellular matrix structure, cell morphology and polarization that enables both path generation and persistent movement.

cell biology↗

bioRxiv2020

Real-Time Multi-Angle Projection Imaging of Biological Dynamics

We introduce a cost-effective and easily implemented scan unit which enables any camera-based microscope to perform projection imaging from diverse viewing angles. We demonstrate this capability on Lattice Light-Sheet and Oblique Plane Microscopy by rapidly delivering projection images with an uncompromised lateral resolution and high optical contrast. By imaging the sample from one or multiple perspectives, our method enables visualization of rapid biological processes, real time stereoscopic imaging as well as three-dimensional particle localization throughout a cellular volume from just two images. Furthermore, because our projection imaging technique provides intuitive three-dimensional renderings in real-time, it improves microscope usability, allows users to more-readily optimize instrument performance and identify biological phenomena of interest on-the-fly, while also reducing data overhead by a factor of >100. We leverage our rapid projection method to image cancer cell morpho-dynamics and calcium signaling in cultured neurons, to perform three-dimensional localization of genetically encoded nanoparticles, as well as to image orthogonal views of an embryonic Zebrafish heart simultaneously.

biophysics↗

bioRxiv2020

Interpretable deep learning of label-free live cell images uncovers functional hallmarks of highly-metastatic melanoma

Deep convolutional neural networks have emerged as a powerful technique to identify hidden patterns in complex cell imaging data. However, these machine learning techniques are often criticized as uninterpretable "black-boxes" - lacking the ability to provide meaningful explanations for the cell properties that drive the machines prediction. Here, we demonstrate that the latent features extracted from label-free live cell images by an adversarial auto-encoding deep convolutional neural network capture subtle details of cell appearance that allow classification of melanoma cell states, including the metastatic efficiency of seven patient-derived xenograft models that reflect clinical outcome. Although trained exclusively on patient-derived xenograft models, the same classifier also predicted the metastatic efficiency of immortalized melanoma cell lines suggesting that the latent features capture properties that are specifically associated with the metastatic potential of a melanoma cell regardless of its origin. We used the autoencoder to generate "in-silico" cell images that amplified the cellular features driving the classifier of metastatic efficiency. These images unveiled pseudopodial extensions and increased light scattering as functional hallmarks of metastatic cells. We validated this interpretation by analyzing experimental image time-lapse sequences in which melanoma cells spontaneously transitioned between states indicative of low and high metastatic efficiency. Together, this data is an example of how the application of Artificial Intelligence supports the identification of processes that are essential for the execution of complex integrated cell functions but are too subtle to be identified by a human expert.

cell biology↗

bioRxiv2020

A Single-Objective Light-Sheet Microscope with 200 nm-Scale Resolution.

We present an Oblique Plane Microscope that uses a bespoke glass-tipped tertiary objective to improve the resolution, field of view, and usability over previous variants. Owing to its high numerical aperture optics, this microscope achieves lateral and axial resolutions that are comparable to the square illumination mode of Lattice Light-Sheet Microscopy, but in a user friendly and versatile format. Given this performance, we demonstrate high-resolution imaging of clathrin-mediated endocytosis, vimentin, the endoplasmic reticulum, membrane dynamics, and Natural Killer-mediated cytotoxicity. Furthermore, we image biological phenomena that would be otherwise challenging or impossible to perform in a traditional light-sheet microscope geometry, including cell migration through confined spaces within a microfluidic device, subcellular photoactivation of Rac1, diffusion of cytoplasmic rheological tracers at a volumetric rate of 14 Hz, and large field of view imaging of neurons, developing embryos, and centimeter-scale tissue sections.

biophysics↗

bioRxiv2019

The nucleus acts as a ruler tailoring cell responses to spatial constraints

The microscopic environment inside a metazoan organism is highly crowded. Whether individual cells can tailor their behavior to the limited space remains unclear. Here, we found that cells measure the degree of spatial confinement using their largest and stiffest organelle, the nucleus. Cell confinement below a resting nucleus size deforms the nucleus, which expands and stretches its envelope. This activates signaling to the actomyosin cortex via nuclear envelope stretch-sensitive proteins, upregulating cell contractility. We established that the tailored contractile response constitutes a nuclear ruler-based signaling pathway involved in migratory cell behaviors. Cells rely on the nuclear ruler to modulate the motive force enabling their passage through restrictive pores in complex three-dimensional (3D) environments, a process relevant to cancer cell invasion, immune responses and embryonic development. One Sentence SummaryNuclear envelope expansion above a threshold triggers a contractile cell response and thus acts as a ruler for the degree of cell deformation.

cell biology↗

bioRxiv2019

Direct Arp2/3-vinculin binding is essential for cell spreading, but only on compliant substrates and in 3D

Cells modify their shape in response to the extracellular environment through dynamic remodeling of the actin cytoskeleton by actin-binding proteins (ABPs) 1-4. The relation between actin dynamics and spreading is well-understood for cells on flat glass coverslips; much less is known about cell morphogenesis in compliant three-dimensional environments, and, in particular, how ABPs contribute to this process 5. Here, we knocked-out a diverse set of ABPs, and evaluated the effect of each on cell spreading on planar glass surfaces (2D) and in reconstituted collagen gels (3D). Our morphometric analyses identify the Arp2/3 complex and its associated regulatory genes among the ABPs that contribute most strongly to cell spreading in 3D, but marginally in 2D. Cells lacking Arp3 have reduced spreading specifically in 3D, and display stiffness-dependent cell-matrix adhesion defects. Through manipulation of vinculin activity, we determine that the Arp3 knock-out phenotype largely arises from the lack of direct interaction between vinculin and Arp2/3 complex. This interaction is dispensable for cell spreading in 2D. These data highlight that actin architectural features necessary for adhesion formation and cell spreading in 3D are efficiently compensated on flat and stiff surfaces.

cell biology↗

bioRxiv2019

A unified role for membrane-cortex detachment during cell protrusion initiation

Cell morphogenesis employs a diversity of membrane protrusions. They are discriminated by differences in force generation. Actin polymerization is the best studied mechanism of force generation, but growing interest in how variable molecular conditions and microenvironments alter morphogenesis has revealed other mechanisms, including intracellular pressure. Here, we show that local depletion of membrane cortex links is an essential step in the initiation of both pressure-based and actin-based protrusions. This observation challenges the quarter-century old Brownian ratchet model of actin-driven membrane protrusion, which requires an optimal balance of actin filament growth and membrane tethering. An updated model confirms membrane-filament detachment is necessary to activate the ratchet mechanism. These findings unify the regulation of different protrusion types, explaining how cells generate robust yet flexible strategies of morphogenesis.

cell biology↗

bioRxiv2018

Cell morphological motif detector for high-resolution 3D microscopy images

Recent advances in light-sheet microscopy enable imaging of cell morphology and signaling with unprecedented detail. However, the analytical tools to systematically measure and visualize the intricate relations between cell morphodynamics, intracellular signaling, and cytoskeletal dynamics have been largely missing. Here, we introduce a set of computer vision and graphics methods to dissect molecular mechanisms underlying 3D cell morphogenesis and to test whether morphogenesis itself affects intracellular signaling. We demonstrate a machine learning based generic morphological motif detector that automatically finds lamellipodia, filopodia, and blebs on various cell types. Combining motif detection with molecular localization, we measure the differential association of PIP2 and KrasV12 with blebs. Both signals associate with bleb edges, as expected for membrane-localized proteins, but only PIP2 is enhanced on blebs. This suggests that local morphological cues differentially organize and activate sub-cellular signaling processes. Overall, our computational workflow enables the objective, automated analysis of the 3D coupling of morphodynamics with cytoskeletal dynamics and intracellular signaling.

cell biology↗