Gaudenz Danuser

Metastasis is a multifaceted process in which cancer cells must overcome diverse biological barriers to establish secondary tumors and drive disease progression. Different cancers exhibit highly non random, organ specific patterns of dissemination, known as organotropism, which arise from cancer cell plasticity and interactions with the host microenvironment. A major challenge in the field is the lack of approaches that capture cancer cell dissemination at the whole organism scale while maintaining physiological relevance. Here, we introduce an in vivo zebrafish model coupled with a data driven computational framework to systematically map and compare patterns of cancer dissemination. Using patient derived Ewing sarcoma cell lines, global analyses reveal pronounced heterogeneity across individual cell lines, which are further resolved by local metrics that capture region specific colonization frequency and cancer cell visit density. This combined global-local approach identifies recurrent spatial preferences across the fish body and uncovers correlated organ targeting, indicating coordinated and structured dissemination dependent on cancer cell line and disease stage. The framework is robust and adaptable to diverse imaging datasets, providing a scalable platform for studying cancer-microenvironment interactions and spatially organized metastatic behavior for various cancer types as well as genetic and drug perturbations, with potential relevance for personalized treatment strategies.

Mesenchymal cell migration starts with the protrusion of the cell’s leading edge, enabled by the branching growth of the actin network. Two crucial molecular players in protrusion are actin filaments and the Arp2/3 complex, the branching agent. Traditionally, protrusion models are intuited from several perturbative experiments. Recent multiplex microscopy imaging data promise to change this paradigm by providing measurements of naturally fluctuating densities of key proteins at the leading edge of unperturbed cells. We report an attempt to reconstruct the mechanistic protrusion model from such data. We analyze fluctuations of densities of actin filaments and Arp2/3 complex and cell edge velocity using phase space and regression analysis to reconstruct linearized dynamics of the coupled actin-Arp2/3-velocity system. We then build a nonlinear model of these dynamics based on previous knowledge of this system and parsimony. The resulting models recover the rates of reactions and mechanical and transport processes in the lamellipodium from a single nonperturbative experiment. The models posit that the dominant nonlinear term in the lamellipodial dynamics corresponds to the retrograde flow of the actin network in the cell frame. The models suggest that the protrusion is dominated by a damped oscillatory cycle resulting from a combination of positive feedback from the Arp2/3 complex to protrusion velocity and negative feedback from actin density to the velocity.

The keratin intermediate filament (IF) cytoskeleton provides mechanical support to load-bearing epithelial cells, but its composition varies depending on cell type and tissue context. This variation, the large size of the keratin protein family, and emerging noncanonical functions of individual keratins suggest that the keratin cytoskeleton may have additional, more complicated roles. We report a surprising interaction between the keratin IF cytoskeleton in epithelial cells and soluble oligomers of vimentin, the principal IF component in mesenchymal cells. Rather than forming a cytoskeletal network, soluble vimentin functions as a recruitment factor, organizing regulatory kinases and nonmuscle myosin on keratin filaments to promote signaling. Soluble vimentin and keratin filaments interact through the intrinsically disordered head domain of each protein. Because vimentin preferentially interacts with keratin 6A-containing filaments, increasing keratin 6A expression, as occurs during skin wound healing and cancer development, triggers this myosin-activating signaling cascade. These results demonstrate how varying composition, soluble pools, and client-scaffold interactions through intrinsically disordered regions allow IFs to organize cellular signals in parallel to their canonical mechanical role, supporting context-dependent cell and tissue regulation.

RhoG is a member of the Rho-family of small GTPases, and is closely related to the canonical Rac1 GTPase, implicated in membrane trafficking, dorsal ruffling, macropinocytosis, and cell protrusion, but its activity has been difficult to visualize directly in living cells with high spatial and temporal resolution. Here, we developed and validated a genetically encoded, single-chain Förster resonance energy transfer (FRET) biosensor for RhoG based on a C-terminal full-length RhoG and an intramolecular RhoG-binding domain derived from ELMO1. The biosensor showed a robust dynamic range when comparing constitutively active and inactive RhoG mutants, responded appropriately to regulation by RhoGDI, GAPs, and GEFs, and detected growth factor–stimulated RhoG activation in live cells. Imaging in mouse embryonic fibroblasts revealed dynamic RhoG activation at leading-edge protrusions, dorsal ruffles, and forming pinocytic and macropinocytic structures. To define the signaling relationship between RhoG and its closely related family member Rac1, we combined the RhoG biosensor with a near-infrared Rac1 FRET biosensor for simultaneous live-cell imaging. Morphodynamic mapping showed that both RhoG and Rac1 activities were positively coupled to edge protrusion, with strongest correlations near the leading-edge, but their direct coupling varied with distance from the edge, indicating partial spatial decoupling within protrusive regions. Inhibition of Src-family kinases altered RhoG dynamics, strongly suppressed Rac1 coupling to protrusion, and inverted the normal positive correlation between RhoG and Rac1 activities. Signaling microdomain analysis further showed that Src inhibition selectively prolonged Rac1 microdomain lifetimes without significantly affecting RhoG domains. Together, these results establish a new biosensor for direct visualization of RhoG activity and reveal that RhoG and Rac1 are coordinated but spatially and temporally distinct components of protrusion-associated signaling networks, with Src-family kinases playing a central role in maintaining their normal coupling.

During development, transplantation, and homeostasis, hematopoietic stem cells (HSCs) migrate to niches to (re)constitute and maintain the hematopoietic system. A solid mechanistic understanding of their movement is essential but remains poorly examined. Here, we used long-term single-cell imaging and tracking to quantify how motility changes as immature HSCs differentiate into multipotent progenitors (MPPs), establishing motility as an early indicator of differentiation. HSCs and early MPPs exhibit rapid, non-directional movement, while more differentiated MPPs move more slowly and directed. Interestingly, ex vivo expanded HSCs retain HSC characteristics with more non-directional movement, suggesting it as a defining HSC trait. Moreover, we show that movement phenotypes can identify cells with higher HSC potential in long-term cultures. HSCs feature a dedicated cytoskeletal architecture characterized by elevated expression of microtubule- and actin-regulating proteins, and by 3D imaging we show that this coincides with reduced cytoskeletal dynamics. FSCN1, a cytoskeletal regulator prevalent in immature cells, controls HSC locomotion by modulating cell shape, and influences cell cycle kinetics and HSC attributes. Finally, THPO, but not SCF or CXCL12, triggers HSC motility through PI3K/AKT signaling.

Cell migration requires the precise coordination of signaling pathways that regulate cytoskeletal dynamics and adhesion turnover. Rho GTPase–activating proteins (RhoGAPs) play critical roles in shaping these processes by controlling the spatial and temporal activity of small GTPases. ARHGAP20 is a RhoA-specific GAP, a downstream target of the Ras-related GTPase Rap1, and has been implicated in cancer cell motility, yet its functional role in coordinating migration-associated signaling remains poorly understood. Here, we investigated the role of ARHGAP20 in cell migration and its impact on the coordination between adhesion- and contractility-associated signaling pathways regulated by Rap1A and RhoA respectively. Using loss-of-function approaches in MTLn3 cells, we show that depletion of ARHGAP20 impairs both directed and random migration, leading to reduced cell velocity and displacement, and increased cell adhesion. To explore the underlying signaling mechanisms, we developed a genetically encoded FRET biosensor to monitor Rap1A activity and combined it with a near-infrared RhoA biosensor to simultaneously analyze their spatiotemporal dynamics in living cells. We found that Rap1A and RhoA activities are negatively coordinated during leading-edge dynamics and that ARHGAP20 depletion enhances this local anticorrelation. To further define where ARHGAP20 regulates Rap1A–RhoA signaling, we applied a microdomain-based analytical framework to quantify local signaling clusters. This analysis revealed that ARHGAP20 selectively modulates Rap1A–RhoA coordination outside focal adhesion regions, while leaving signaling correlations and overlap within focal adhesions unchanged. Subcellular localization analyses further reveal that ARHGAP20 is largely excluded from focal adhesions but associates with microtubules, the endoplasmic reticulum, the Golgi apparatus, and multiple Rab-positive vesicular compartments, supporting a trafficking-dependent mechanism for its spatial targeting. Together, our results identify ARHGAP20 as a regulator of cell migration that modulates the coordination between Rap1A and RhoA signaling through intracellular vesicular trafficking, highlighting the GAP’s role in organizing the spatial coupling between signaling pathways in adhesion-associated regions required for efficient cell migration.

The tissue-resident immune system involves complex 3D assemblies that interact with extended structures such as blood vessels and nerves. These interactions are difficult to study using conventional 2D profiling because they span many tissue sections. In animal tissues, volumetric imaging approaches such as light-sheet fluorescence microscopy (LSFM) are widely used to study 3D tissue organization, with labelling often aided by genetically encoded reporters and vascular dyes. In contrast, LSFM of human specimens remains underdeveloped because most clinical samples are available only as formalin-fixed paraffin-embedded (FFPE) tissue, limiting labeling strategies primarily to dyes and antibodies. Here, we present a volumetric cyclic immunofluorescence (v-CyCIF) and virtual H&E toolbox that overcomes key barriers to multiplexed imaging of immune cells and nerves in human specimens up to 1 mm thick. We use v-CyCIF to study neuroimmune interactions in normal and cancer tissues and to immunoprofile intact secondary and tertiary lymphoid structures. Re-embedding and sectioning of specimens following volumetric imaging enables high-plex high-resolution analysis of subcellular structures and cell-cell interactions associated with immune cell activity. v-CyCIF therefore provides a flexible framework for multi-scale 3D profiling of clinical specimens across imaging formats and resolutions.

Fluorescent biosensors that report protein conformation in vivo have been invaluable for understanding how the spatio-temporal dynamics of signaling controls cells. However, for GTPases these biosensors report the activated conformation using reagents that block the binding of downstream proteins, generating dominant negative effects and altering normal cell physiology. We present here a generalizable design to make GTPase biosensors (AlloRac1 and AlloCdc42), in which a circularly permuted fluorescent protein is inserted into a conserved loop allosterically connected to the effector binding site, generating activity-dependent fluorescence without blocking ligand interactions. The Rac1 biosensor showed that effector interactions led to increased Rac1 activation, indicating an auto-regulatory positive feedback made visible by the new biosensor design. This feedback regulated the kinetics and localization of Rac1 activity, including Rac1 activity gradients that controlled motility. Feedback was generated through Rac1 interaction with the effector Pak1, which led to further activation of Rac1 by the guanine exchange factor β-Pix. The new biosensor approach enables quantitative imaging of previously obscure spatio-temporal dynamics in GTPase regulation.

For over three decades, we have known that oncogenic RAS alters the actin cytoskeleton organization and cell surface morphology ^1,2 . RAS activates the GTPase RAC1, which triggers the growth of branched actin networks to promote cell membrane protrusions ^3,4 . In melanoma, the hyperactive RAC1 mutant, Rac1 ^P29S , was recently shown to drive extended lamellipodia, which then empower cell proliferation through sequestration and localized inhibition of the merlin tumor suppressor ⁵ . This discovery illustrates cell morphological programs not only as outputs but also as regulators of human oncogenic signals. Hence, we wondered whether the pronounced branched actin-driven membrane protrusions (BAMPs) downstream of oncogenic RAS are not mere outputs of RAS signaling but rather an active component in mediating the oncogenic penetrance of RAS mutants. We used volumetric light sheet microscopy and biochemical approaches to investigate the role of BAMPs in regulating the molecular signaling of oncogenic KRAS in pancreatic and lung cancer models. We found that elevated BAMP formation regulated the interaction of oncogenic KRAS with downstream effectors, specifically with the RAC1 GEF TIAM1. This implies that BAMPs amplify their own upstream regulators in a positive feedback. This meritorious cycle upregulates cyclin D1 expression by inactivating the merlin tumor suppressor, independently of the mitogen activated protein kinase pathway (MAPK). In the absence of BAMPs, cells carrying oncogenic KRAS mutations are unable to attain their full penetrance in proliferation. Overall, this work unveils the long-overlooked role of branched actin-driven cell morphology in the functionalization of KRAS mutants as potent oncogenes.