Biology subjects

Weigert, R.

Publications and source records attributed to Weigert, R..

13 recordsLinked to original sources

bioRxiv2026

Tunneling nanotubes mediate mitochondrial homeostasis in cancer

Tumor progression is driven by cancer cells ability to establish a cellular network through tunneling nanotube-like connections (TNTs), which enable mitochondrial exchange both within the tumor cells and with the tumor microenvironment (TME). However, the effects of mitochondrial transfer between tumor and non-tumor cells, and its occurrence in vivo, remain poorly understood. In this study, we demonstrate bidirectional mitochondrial transfer: damaged mitochondria from Glioblastoma (GBM) cells trigger mitophagy in non-tumoral astrocytes (AS), while healthy mitochondria from AS enhance the metabolic activity of GBM cells. Furthermore, intravital subcellular microscopy (ISMic) in a live animal model, allowed the visualization of TNT connections with characteristics similar to those observed in vitro and supported TNT-mediated mitochondrial transfer in vivo. These findings provide critical insights into TME interactions and their contribution to cancer resilience, highlighting TNTs as a potential target for therapeutic intervention and paving the way for further research into their role in cancer.

Cancer Biology↗

bioRxiv2026

Spatially Distinct Myosin II Architectures Regulate Protrusion Dynamics and Directional Persistence during Immune Cell Migration

Directional persistence is essential for efficient immune cell migration in tissues, yet how cytoskeletal systems stabilize migration in complex three-dimensional environments remains unclear. Using intravital subcellular microscopy and quantitative analysis of membrane dynamics, we identify two spatially distinct architectures of non-muscle myosin II (NMII) that coordinate protrusion dynamics during neutrophil migration. In vivo and in collagen matrices, NMII assembles at the leading edge into lattice-like structures that are structurally and functionally distinct from rear contractile actomyosin bundles. Protrusion-resolved analyses reveal that directional persistence correlates strongly with protrusion lifetime and sustained NMII engagement, with rear NMII load showing the strongest association with protrusion persistence. Strikingly, directional migration is not determined by the abundance of favorable protrusions but by their temporal organization during migration. Pharmacological perturbations that redistribute NMII activity disrupt this temporal organization and alter migration trajectories. Together, these findings reveal that spatially distinct NMII architectures coordinate protrusion dynamics across time to stabilize directional migration in complex environments. HighlightsO_LIMigrating neutrophils assemble spatially distinct myosin II architectures at the leading edge and rear C_LIO_LIProtrusion dynamics and directional persistence are linked to sustained myosin II engagement C_LIO_LIDirectional migration emerges from the temporal organization of protrusion states rather than their abundance C_LI

cell biology↗

bioRxiv2026

Intravital single-molecule imaging reveals cytoskeletal turnover as a driver of membrane remodeling in live animals

Understanding how cells regulate plasma membrane architecture inside intact living organs in a live animal has been limited by the inability to directly measure molecular dynamics in vivo. Here we introduce intravital single-molecule microscopy (iSiMM), an imaging approach that enables tracking of individual, endogenously expressed cytoskeletal components at the plasma membrane in live mice. Applying iSiMM to murine acinar secretory cells, we identify discrete basolateral membrane domains built on deeply folded membrane infolds that function as a pre-existing membrane reservoir. Single-molecule measurements reveal continuous, regulated molecular turnover within these domains. Physiological stimulation accelerates cytoskeletal exchange promoting rapid membrane unfolding and cell expansion. Together, these findings establish iSiMM as a general strategy for probing molecular kinetics underlying dynamic cellular behaviors in intact organs. One-sentence summaryIntravital single-molecule microscopy enables direct measurement of molecular kinetics underlying dynamic cellular behaviors in intact living organs.

cell biology↗

bioRxiv2025

Longitudinal Imaging of the Premalignant Tumor Microenvironment Reveals Transient Myeloid States Predictive of Tumor Fate

The spontaneous regression of cancer lesions illustrates the power of immune surveillance; yet these transient events have largely escaped systematic analysis. Using longitudinal intravital microscopy in a carcinogen-induced model of head and neck cancer, we followed premalignant lesions within the same animals for 24 weeks at single-cell resolution. This strategy uncovered three trajectories: progression, stability, or regression, and enabled direct analysis of immune dynamics underlying each fate. Lesion outcome was determined by the spatial organization of myeloid-derived antigen-presenting cells: regressing lesions were characterized by dense clusters of myeloid-derived cells associated with CXCL9+/CXCL10+ expression and T cell recruitment, whereas progressing lesions displayed a scattered infiltration of these cells. Remarkably, transient myeloid clusters arose prior to any detectable lesion formation and marked regions that would later develop into premalignant lesions. These findings identify spatiotemporal myeloid organization as an early determinant of tumor fate and provide a mechanistic framework for predicting and intercepting cancer at its inception. Summary SentenceEarly myeloid architecture dictates cancer fate: dense CXCL9/CXCL10 clusters with T-cell enrichment accompany regression, whereas sparse infiltration predicts progression. Transient pre-lesional myeloid clusters emerge at future tumor sites, revealing immune organization as an early determinant of malignancy.

cancer biology↗

bioRxiv2025

Dissection of Gαs and Hedgehog signaling crosstalk reveals therapeutic opportunities to target adenosine receptor 2b in Hedgehog-dependent tumors

Basal cell carcinoma (BCC), the most common human cancer, is driven by hyperactivation of Hedgehog Smoothened (SMO) and GLI transcription. Gs and protein kinase A (PKA) negatively regulate Hedgehog signaling, offering an alternative BCC development and treatment pathway. Here, using histology alongside bulk and single-cell RNA sequencing, we find that mouse BCC-like tumors that originate from Gs pathway inactivation are strikingly similar to those driven by canonical Hedgehog SMO. Interestingly, mutations that reduce Gs and PKA activity are present in human BCC. Tumors from Gs pathway inactivation are independent of the canonical Hedgehog regulators SMO and GPR161, establishing them as an SMO-independent oncogenic Hedgehog signaling model. Finally, we demonstrate that activation of the Gs-coupled adenosine 2B receptor counteracts oncogenic SMO, reducing Hedgehog signaling and tumor formation and offering a potential therapeutic strategy for BCC.

cancer biology↗

bioRxiv2024

Control of Epithelial Tissue Organization by mRNA Localization

mRNA localization to specific subcellular regions is common in mammalian cells but poorly understood in terms of its physiological roles1-6,7. This study demonstrates the functional importance of Net1 mRNA, which we find prominently localized at the dermal-epidermal junction (DEJ) in stratified squamous epithelia. Net1 mRNA accumulates at DEJ protrusion-like structures that interact with the basement membrane and connect to a mechanosensitive network of microfibrils. Disrupting Net1 mRNA localization in mouse epithelium alters DEJ morphology and keratinocyte-matrix connections, affecting tissue homeostasis. mRNA localization dictates Net1 protein distribution and its function as a RhoA GTPase exchange factor (GEF). Altered RhoA activity is in turn sufficient to alter the ultrastructure of the DEJ. This study provides a high-resolution in vivo view of mRNA targeting in a physiological context. It further demonstrates how the subcellular localization of a single mRNA can significantly influence mammalian epithelial tissue organization, thus revealing an unappreciated level of post-transcriptional regulation that controls tissue physiology.

cell biology↗

bioRxiv2023

Spatial and Temporal Coordination of Force-generating Actin-based Modules Drives Membrane Remodeling In Vivo

Membrane remodeling drives a broad spectrum of cellular functions, and it is regulated through mechanical forces exerted on the membrane by cytoplasmic complexes. Here, we investigate how actin filaments dynamically tune their structure to control the active transfer of membranes between cellular compartments with distinct compositions and biophysical properties. Using intravital subcellular microscopy in live rodents we show that: a lattice composed of linear filaments stabilizes the granule membrane after fusion with the plasma membrane; and a network of branched filaments linked to the membranes by Ezrin, a regulator of membrane tension, initiates and drives to completion the integration step. Our results highlight how the actin cytoskeleton tunes its structure to adapt to dynamic changes in the biophysical properties of membranes.

cell biology↗

bioRxiv2023

Fusion-Negative Rhabdomyosarcoma xenografts in murine tongue provide a model for the study of local invasion, intravasation and metastasis at cellular and subcellular levels in live animals

PAX3/7 Fusion-negative rhabdomyosarcoma (FN-RMS) is a childhood mesodermal lineage malignancy with a poor prognosis for metastatic or relapsed cases. Towards achieving a more complete understanding of advanced FN-RMS, we developed an orthotopic tongue xenograft model for studies of molecular basis of FN-RMS invasion and metastasis. The behavior of FN-RMS cells injected into murine tongue was examined using in vivo bioluminescence imaging, non-invasive intravital microscopy (IVM), and histopathology and compared to the prevailing hindlimb intramuscular and subcutaneous xenografts. FN-RMS cells were retained in the tongue and invaded locally into muscle mysial spaces and vascular lumen. While evidence of hematogenous dissemination to the lungs occurred in tongue and intramuscular xenografts, evidence of local invasion and lymphatic dissemination to lymph nodes only occurred in tongue xenografts. IVM and RNA-seq of tongue xenografts reveal shifts in cellular phenotype and differentiation state in tongue xenografts. IVM also shows homing to blood and lymphatic vessels, lymphatic intravasation, and dynamic membrane protrusions. Based on these findings, the tongue orthotopic xenograft of FN-RMS is a valuable model for tumor progression studies at the tissue, cellular and subcellular levels providing insight into kinetics and molecular bases of tumor invasion and metastasis and, hence, new therapeutic avenues for advanced FN-RMS.

cancer biology↗

bioRxiv2023

Growth factor-induced desialylation for the fast control of endocytosis

It is commonly assumed that the glycan makeup of glycoproteins that reach the cell surface is final and static. Here, we challenge this notion by the discovery of a molecular switch that induces acute and reversible changes of glycans on the plasma membrane. We demonstrate that within minutes, the epidermal growth factor triggers the galectin-driven endocytosis of cell surface glycoproteins, such as integrins, that are key regulators of cell adhesion and migration. The onset of this process, mediated by the Na+/H+ antiporter NHE-1 and the neuraminidases Neu1/3, requires the pH-triggered enzymatic removal of sialic acids whose presence otherwise prevents galectin binding. Desialylated glycoproteins are then retrogradely transported to the Golgi apparatus where their glycan makeup is reset, and their function is repurposed to regulate EGF-dependent invasive cell migration. Glycosylation at the cell surface thereby emerges as a dynamic and reversible regulatory post-translational modification that controls a highly adaptable trafficking pathway.

cell biology↗

bioRxiv2022

GATA transcription factors drive initial Xist upregulation after fertilization through direct activation of a distal enhancer element

To ensure dosage compensation for X-linked genes between the sexes, one X chromosome is silenced during early embryonic development of female mammals. This process of X-chromosome inactivation (XCI) is initiated through upregulation of the RNA Xist from one X chromosome shortly after fertilization. Xist then mediates chromosome-wide gene silencing in cis and remains expressed in all cell types except the germ line and the pluripotent state, where XCI is reversed. The factors that drive Xist upregulation and thereby initiate XCI remain however unknown. We identify GATA transcription factors as potent Xist activators and demonstrate that they are essential for the activation of Xist in mice following fertilization. Through a pooled CRISPR activation screen we find that GATA1 can drive ectopic Xist expression in murine embryonic stem cells (mESCs). We demonstrate that all GATA factors can activate Xist directly via a GATA-responsive regulatory element (RE79) positioned 100 kb upstream of the Xist promoter. Additionally, GATA factors are essential for the induction of XCI in mouse preimplantation embryos, as simultaneous deletion of three members of the GATA family (GATA1/4/6) in mouse zygotes effectively prevents Xist upregulation. Thus, initiation of XCI and possibly its maintenance in distinct lineages of the preimplantation embryo is ensured by the combined activity of different GATA family members, and the absence of GATA factors in the pluripotent state likely contributes to X reactivation. We thus describe a form of regulation in which the combined action of numerous tissue-specific factors can achieve near-ubiquitous expression of a target gene.

genetics↗

bioRxiv2019

A role for keratins in supporting mitochondrial organization and function in skin keratinocytes

Mitochondria fulfill essential roles in ATP production, metabolic regulation, calcium signaling, generation of reactive oxygen species (ROS) and additional determinants of cellular health. Recent studies have highlighted a role for mitochondria during cell differentiation, including in skin epidermis. The observation of oxidative stress in keratinocytes from Krt16 null mouse skin, a model for pachyonychia congenita (PC)-associated palmoplantar keratoderma, prompted us to examine the role of Keratin (K) 16 protein and its partner K6 in regulating the structure and function of mitochondria. Electron microscopy revealed major anomalies in mitochondrial ultrastructure in late stage, E18.5, Krt6a/Krt6b null embryonic mouse skin. Follow-up studies utilizing biochemical, metabolic, and live imaging readouts showed that, relative to controls, skin keratinocytes null for Krt6a/Krt6b or Krt16 exhibit elevated ROS, reduced mitochondrial respiration, intracellular distribution differences and altered movement of mitochondria within the cell. These findings highlight a novel role for K6 and K16 in regulating mitochondrial morphology, dynamics and function and shed new light on the causes of oxidative stress observed in PC and related keratin-based skin disorders.

cell biology↗

bioRxiv2019

The LTB4-BLT1 signaling axis coordinates actomyosin dynamics and β-2 Integrin trafficking to drive intravascular neutrophil response to infection

The eicosanoid Leukotriene B4 (LTB4) relays chemotactic signals to direct neutrophil migration to inflamed sites through its receptor BLT1. However, the mechanisms by which the LTB4-BLT1 axis relays chemotactic signals during intravascular neutrophil response to inflammation remain unclear. Here, we report that LTB4 produced by neutrophils acts as an autocrine/paracrine signal to direct the vascular recruitment, arrest and extravasation of neutrophils in a sterile inflammation model in the mouse footpad. Using Intravital Subcellular Microscopy (ISMic), we reveal that LTB4 elicits sustained cell polarization and adhesion responses during neutrophil arrest in vivo. Specifically, LTB4 signaling coordinates the dynamic redistribution of non-muscle Myosin IIA (NMIIA) and {beta}2-integrin (Itgb2), which facilitate neutrophil arrest and extravasation. Notably, we also found that neutrophils shed extracellular vesicles (EVs) in the vascular lumen, and that inhibition of EV release blocks LTB4-mediated autocrine/paracrine signaling required for neutrophil arrest and extravasation. Overall, we uncover a novel complementary mechanism by which LTB4 relays extravasation signals in neutrophils during early inflammation response. SUMMARYNeutrophils arrest and extravasate from the blood vessels in response to infection and injury. Using intravital subcellular microscopy, Subramanian et al. identify a role for extracellular vesicles-based autocrine/paracrine LTB4-BLT1 signaling in promoting the re-arrangement of actomyosin cytoskeleton and {beta}2-integrin during neutrophil extravasation in live animals.

cell biology↗

bioRxiv2018

Cdc42 negatively regulates endocytosis during apical plasma membrane maintenance and development in mouse tubular organs in vivo

Lumen establishment and maintenance are fundamental for tubular organs physiological functions. Most of the studies investigating the mechanisms regulating this process have been carried out in cell cultures or in smaller organisms, whereas little has been done in mammalian model systems in vivo. Here we used the salivary glands of live mice to examine the role of the small GTPase Cdc42 in the regulation of the homeostasis of the intercellular canaliculi, a specialized apical domain of the acinar cells, where protein and fluid secretion occur. Depletion of Cdc42 in adult mice induced a significant expansion of the apical canaliculi, whereas depletion at late embryonic stages resulted in a complete inhibition of their post-natal formation. In addition, intravital subcellular microscopy revealed that reduced levels of Cdc42 affected membrane trafficking from and towards the plasma membrane, highlighting a novel role for Cdc42 in membrane remodeling through the negative regulation of selected endocytic pathways.

cell biology↗