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Biology subjects

Fischer, R. S.

Publications and source records attributed to Fischer, R. S..

5 recordsLinked to original sources

Remodeling of cholesterol metabolism in melanoma progression regulates nuclear membrane mechanics.

Metastatic cancer cells migrate through physically restrictive environments where nuclear deformation ruptures the nuclear envelope (NE) resulting in genomic instability. Although structural proteins of the NE are established regulators of nuclear mechanics, how nuclear membrane (NM) lipid composition contributes to its biophysical response to mechanical stress remains poorly understood. Here, we show that melanoma progression is associated with broad transcriptional remodeling of cholesterol homeostasis that is established early in disease progression, with altered expression of cholesterol biosynthesis genes associated with reduced patient survival. Building on our previous identification of the inner NM sterol reductase lamin B receptor (LBR) as a regulator of NE fragility, we find that LBR-dependent cholesterol biosynthesis promotes cholesterol enrichment within the NM and alters its spatial organization during cellular confinement. Quantitative fluorescence and lifetime imaging of biosensors of cholesterol distribution, lipid order, and tension reveals that cholesterol remodeling alters NM lipid organization and results in high NM tension in melanoma cells that is selectively reduced by cholesterol depletion. Cellular confinement generates nuclear blebs where cholesterol becomes preferentially enriched at highly curved, rupture-prone regions of the NM. Together, our results establish cholesterol-dependent NM remodeling as a regulator of nuclear mechanics and link metabolic changes acquired during cancer progression to the biophysical response of the NM to mechanical stress.

cell biology↗

Observing biological spatio-angular structures and dynamics with statistical image reconstruction and polarized fluorescence microscopy

Understanding molecular orientation and density distributions is essential for exploring biological structure and function. Polarized fluorescence microscopy (PFM) provides insights into molecular architecture but struggles to resolve three-dimensional (3D) molecular orientation distributions, particularly in densely labeled or structurally complex specimens. To address this, we introduce the efficient generalized Richardson-Lucy (eGRL) algorithm, a robust framework for reconstructing 3D molecular density and orientation (spatio-angular) distributions from PFM data. By modeling the imaging process in spatio-angular hyperspace, we propose a maximum-likelihood solution enhanced by dimensionality reduction and angular domain transformation to overcome computational challenges. eGRL improves accuracy and efficiency across different PFM implementations, enabling use on standard platforms. We utilize our methods to resolve biological spatio-angular structures and dynamics otherwise impossible to resolve, including the tangential alignment of actin filaments in U2OS cells, nanowire-guided cytoskeletal organization in NIH3T3 cells, rotational actin patterns in live HeLa protrusions, and membrane tension-induced anisotropy in live macrophages.

bioengineering↗

Three-dimensional spatio-angular fluorescence microscopy with a polarized dual-view inverted selective-plane illumination microscope (pol-diSPIM)

Polarized fluorescence microscopy is a valuable tool for measuring molecular orientations, but techniques for recovering three-dimensional orientations and positions of fluorescent ensembles are limited. We report a polarized dual-view light-sheet system for determining the three-dimensional orientations and diffraction-limited positions of ensembles of fluorescent dipoles that label biological structures, and we share a set of visualization, histogram, and profiling tools for interpreting these positions and orientations. We model our samples, their excitation, and their detection using coarse-grained representations we call orientation distribution functions (ODFs). We apply ODFs to create physics-informed models of image formation with spatio-angular point-spread and transfer functions. We use theory and experiment to conclude that light-sheet tilting is a necessary part of our design for recovering all three-dimensional orientations. We use our system to extend known two-dimensional results to three dimensions in FM1-43-labelled giant unilamellar vesicles, fast-scarlet-labelled cellulose in xylem cells, and phalloidin-labelled actin in U2OS cells. Additionally, we observe phalloidin-labelled actin in mouse fibroblasts grown on grids of labelled nanowires and identify correlations between local actin alignment and global cell-scale orientation, indicating cellular coordination across length scales.

biophysics↗

Lamin B Receptor Upregulation in Metastatic Melanoma Causes Cholesterol-Mediated Nuclear Envelope Fragility

Metastatic cancer cells migrate through regions of tissue confinement, causing nuclear envelope (NE) rupture and heritable DNA damage. We discovered that cells from multiple cancers have increased NE fragility in confinement and transcriptional upregulation of nuclear genes compared to benign counterparts. A bioinformatic-driven siRNA screen revealed that lamin B receptor (LBR) upregulation correlates with melanoma progression and NE fragility. Increased LBR cholesterol synthase activity causes accumulation of cholesterol in the NE, which is necessary and sufficient for nuclear deformability and NE rupture in cells confined in vitro and is associated with NE rupture in invasive cells migrating out of tumor organoids and tumors in vivo. Thus, LBR upregulation causes excess NE cholesterol, driving nuclear fragility in confined migrating melanoma cells, establishing a direct role for nuclear membrane lipid composition in metastatic cancers.

cell biology↗

Characterization of Peripheral Blood Mononuclear Cells Gene Expression Profiles of Pediatric Staphylococcus aureus Persistent and Non-Carriers Using a Targeted Assay

Defects in innate immunity affect many different physiologic systems and several studies of patients with primary immunodeficiency disorders demonstrated the importance of innate immune system components in disease prevention or colonization of bacterial pathogens. To assess the role of the innate immune system on nasal colonization with Staphylococcus aureus, innate immune responses in pediatric S. aureus nasal persistent carriers (n=15) and non-carriers (n=15) were profiled by analyzing co-clustered gene sets (modules) identified through large-scale transcriptome data analysis as the basis for the development of a targeted assay. We stimulated previously frozen peripheral blood mononuclear cells (PBMCs) from these subjects with i) a panel of TLR ligands, ii) live S. aureus (either a mixture of strains or stimulation with respective carriage isolates), or iii) heat-killed S. aureus. We found no difference in responses between carriers and non-carriers when PBMCs were stimulated with a panel of TLR ligands. However, PBMCs stimulated with live S. aureus elicited a significantly different response that also differed from the response elicited following stimulation with dead S. aureus. Furthermore, we observed a distinct stimulation profile for PBMCs isolated from persistent carriers stimulated with their respective live or dead carriage strains compared to responses observed for PBMCs isolated from non-carriers that were similar regardless of whether or not the bacteria were alive or not. These data suggested that innate pathway signaling is different between persistent and non-carriers of S. aureus.

immunology↗