Biology subjects

Sawicka, K.

Publications and source records attributed to Sawicka, K..

7 recordsLinked to original sources

bioRxiv2026

A three-dimensional imaging pipeline for the detection of vasculogenic mimicry in mouse tumor models

Vasculogenic mimicry (VM) describes the ability for tumor cells to mimic endothelial cells and form tumor-lined blood vessels. Although VM is associated with poor prognosis, resistance to therapeutics and metastasis, its presence, relevance and functionality have been debated, largely due to the techniques employed to determine its presence. Dual CD31 and periodic acid-Schiff (PAS) histological staining, the method widely used to detect VM within tumor tissue, is hampered by issues with artifacts of 2D sections and does not assess active blood flow through vessels and hence cannot confirm vessel functionality. Here, we present a 3D imaging pipeline to detect functionally perfused VM and host vessels within mouse tumor models. Mice tumor models are intravenously injected with fluorescently-labelled tomato lectin which binds to the lumen of functional blood vessels. These tumors are thickly sliced, optically-cleared, stained with endothelial markers and imaged using light-sheet microscopy. We apply this imaging modality to image tumors from six breast cancer patient-derived tissue xenograft models, an orthotopic mammary cancer model and two small cell lung cancer circulating-tumor cell explant models, where VM vessels, mosaic vessels and host-VM vessel junctions can be detected. To our knowledge, this is the largest collection of 3D VM vessel images to date, providing convincing evidence of the presence and functionality of VM within tumors and defining the level of evidence necessary to detect VM in vivo.

cancer biology↗

bioRxiv2021

WILD-seq: Clonal deconvolution of transcriptomic signatures of sensitivity and resistance to cancer therapeutics in vivo.

Tumor heterogeneity is thought to be a major barrier to successful cancer treatment due to the presence of drug resistant clonal lineages. However, identifying the characteristics of such lineages that underpin resistance to therapy has remained challenging. Here we utilize clonal transcriptomics with WILD-seq; Wholistic Interrogation of Lineage Dynamics by sequencing, in mouse models of triple-negative breast cancer (TNBC) to understand response and resistance to therapy, including BET bromodomain inhibition and taxane-based chemotherapy. This analysis revealed oxidative stress protection by NRF2 as a major mechanism of taxane resistance and led to the discovery that our tumor models are collaterally sensitive to asparagine deprivation therapy using the clinical stage drug L-asparaginase after frontline treatment with docetaxel. In summary, clonal transcriptomics with WILD-seq identifies mechanisms of resistance to chemotherapy that are also operative in patients and pin points asparagine bioavailability as a druggable vulnerability of taxane resistant lineages.

cancer biology↗

bioRxiv2021

FMRP regulates mRNAs encoding distinct functions in the cell body and dendrites of CA1 pyramidal neurons

Neurons are believed to rely on dendritic localization and translation of mRNAs in order to generate activity-dependent changes in the synaptic plasticity. Here, we develop a strategy combining compartment-specific CLIP and TRAP in conditionally tagged mice to precisely define the ribosome-bound dendritic transcriptome of CA1 pyramidal neurons. This revealed transcripts that have differentially localized alternative 3UTR and splicing isoforms. FMRP targets are overrepresented among dendritic mRNAs, and compartment-specific FMRP-CLIP defined 383 dendritic FMRP targets, and also allowed for segregation of whole-cell FMRP targets into functional modules that are locally regulated by FMRP. In the absence of FMRP, dendritic FMRP targets show increased ribosome association, consistent with reported roles for FMRP in translational repression. Together, the data support a model in which distinct patterns of FMRP localization allow it to differentially regulate the expression of nuclear proteins and synaptic proteins within different compartments of a single neuronal cell type.

neuroscience↗

bioRxiv2019

FMRP binding to a ranked subset of long genes is revealed by coupled CLIP and TRAP in specific neuronal cell types

Loss of function of the Fragile X Mental Retardation Protein (FMRP) in human Fragile X Syndrome (FXS) and in model organisms results in phenotypes of abnormal neuronal structure and dynamics, synaptic function and connectivity which may contribute to a state of neuronal, circuit and organism hyperexcitability. Previous in vivo identification of FMRP association with specific mRNA targets in mouse brain revealed that FMRP regulates the translation of a large fraction of the synaptic proteome in both pre- and post-synaptic compartments as well as many transcription factors and chromatin modifying proteins. However, it was not previously possible to determine the ratio of FMRP binding to transcript abundance due to the complexity of different neuronal cell types in whole brain. Moreover, it has been difficult to link the translational regulation of specific targets to model phenotypes or human symptoms. For example, loss-of-function of FMRP in the Purkinje cells of the cerebellum results in three cell autonomous phenotypes related to learning and memory, including enhanced mGluR-LTD at parallel fiber synapses, altered dendritic spines and behavioral deficits in a eyeblink-conditioning learning paradigm shared by human FXS patients. The molecular basis for these and related human Fragile X phenotypes is unknown. To address these critical issues we have developed a new mouse model (the Fmr1 cTAG mouse) in which endogenous FMRP can be conditionally tagged for RNA:protein crosslinking and immunoprecipitation (CLIP) identification of the RNAs with which it interacts in vivo. We used the Fmr1 cTAG mouse to quantitatively evaluate FMRP-mRNA association in Purkinje and cerebellar granule neurons which together comprise the parallel-fiber synapse. We calculated a stoichiometrically ranked list of FMRP RNA binding events by normalizing to ribosome-associated transcript abundance determined by TRAP-seq, and now definitively find that FMRP associates with specific sets of mRNAs which differ between the two cell types. In Purkinje cells, many components of the mGluR signaling pathway are FMRP targets including the top-ranked Purkinje cell mRNA Itpr1, encoding the IP3 receptor, the function of which is critical to proper mGluR-dependent synaptic plasticity. In sum, this novel approach provides the first ranked list of FMRP target mRNAs and further reveals that FMRP regulates a specific set of long neural genes related to relevant cell autonomous phenotypes.\n\nHighlightsO_LIWe have created a mouse model in which endogenous FMRP can be conditionally tagged.\nC_LIO_LIUsing tag-specific CLIP we describe ranked and specific sets of in vivo FMRP mRNA targets in two types of neurons.\nC_LIO_LIThis ranking was used to reveal that FMRP regulates mRNAs with long coding sequences.\nC_LIO_LIFMRP mRNA targets in Purkinje cells, including the top-ranked IP3 receptor, are related to cell-autonomous Fragile X phenotypes.\nC_LIO_LIWe have updated our previous list of whole mouse brain FMRP mRNA targets with more replicates, deeper sequencing and improved analysis\nC_LIO_LIThe use of tagged FMRP in less abundant cell populations allowed identification of novel mRNA targets missed in a whole brain analysis\nC_LI

neuroscience↗

bioRxiv2018

FMRP association with and regulation of Fragile X granules exhibit circuit-dependent requirements for the KH2 RNA binding domain

The localization and translation of mRNAs is controlled by a diverse array of ribonucleoprotein particles (RNPs), multimolecular complexes containing mRNAs and RNA binding proteins. Fragile X granules (FXGs) are a family of RNPs that exemplify the diversity of RNA granules in the mammalian nervous system. FXGs are found in a conserved subset of neurons, where they localize exclusively to the axonal compartment. Notably, the specific RNA binding proteins and mRNAs found in FXGs depend on brain circuit and neuron type, with all forebrain FXGs containing Fragile X mental retardation protein (FMRP), the protein mutated in the human autism-related disorder Fragile X syndrome. FMRP negatively regulates FXG abundance but is not required for their association with ribosomes or mRNA. To better understand the circuit-dependent mechanisms whereby FMRP associates with and regulates FXGs, we asked how a disease-causing point mutation, I304N, in the KH2 RNA binding domain of FMRP affects these granules in two brain regions - cortex and hippocampus. We found that FMRPI304N had a reduced association with FXGs, as it was absent from approximately half of FXGs in cortex and nearly all FXGs in hippocampus. FXG abundance correlated with the number of FMRP-containing FXGs, suggesting that FMRP regulates FXG abundance by KH2-independent mechanisms that occur locally within the granules. Together, these findings illustrate that cell type-dependent mechanisms guide the assembly of similar RNA granules. Further, point mutations in RNA granule components may lead to cell type-dependent phenotypes that produce atypical forms of disorders that normally arise from more severe mutations.

neuroscience↗

bioRxiv2018

Whole-genome deep learning analysis reveals causal role of noncoding mutations in autism

We address the challenge of detecting the contribution of noncoding mutations to disease with a deep-learning-based framework that predicts specific regulatory effects and deleterious disease impact of genetic variants. Applying this framework to 1,790 Autism Spectrum Disorder (ASD) simplex families reveals autism disease causality of noncoding mutations by demonstrating that ASD probands harbor transcriptional (TRDs) and post-transcriptional (RRDs) regulation-disrupting mutations of significantly higher functional impact than unaffected siblings. Importantly, we detect this significant noncoding contribution at each level, transcriptional and post-transcriptional, independently and after multiple hypothesis correction. Further analysis suggests involvement of noncoding mutations in synaptic transmission and neuronal development, and reveals a convergent genetic landscape of coding and noncoding (TRD and RRD) de novo mutations in ASD. We demonstrate that sequences carrying prioritized proband de novo mutations possess transcriptional regulatory activity and drive expression differentially, and highlight a link between noncoding mutations and IQ heterogeneity in ASD probands. Our predictive genomics framework illuminates the role of noncoding mutations in ASD, prioritizes high impact transcriptional and post-transcriptional regulatory mutations for further study, and is broadly applicable to complex human diseases.

genomics↗

bioRxiv2018

ZFP36 RNA-binding proteins restrain T-cell activation and anti-viral immunity

Dynamic post-transcriptional control of RNA expression by RNA-binding proteins (RBPs) is critical during immune response. ZFP36 RBPs are prominent inflammatory regulators linked to autoimmunity and cancer, but functions in adaptive immunity are less clear. We used HITS-CLIP to define ZFP36 targets in T-cells, which revealed unanticipated actions in regulating T-cell activation, proliferation, and effector functions. Transcriptome and ribosome profiling showed that ZFP36 represses mRNA target abundance and translation, notably through a novel class of AU-rich sites in coding sequence. Functional studies revealed that ZFP36 regulates early T-cell activation kinetics in a cell autonomous manner, by attenuating activation marker expression, limiting T-cell expansion, and promoting apoptosis. Strikingly, loss of ZFP36 in vivo accelerated T-cell responses to acute viral infection, and enhanced anti-viral immunity. These findings uncover a critical role for ZFP36 RBPs in restraining T-cell expansion and effector functions, and suggest ZFP36 inhibition as a novel strategy to enhance immune-based therapies.

molecular biology↗