Biology subjects

Scarcelli, G.

Publications and source records attributed to Scarcelli, G..

10 recordsLinked to original sources

bioRxiv2025

Polarization Increases Nuclear Stiffness in Macrophages Despite Reduction in Lamin A/C Levels

Macrophages are innate immune cells contributing to tissue homeostasis and various pathologies. Signals from their environment can lead macrophages to adapt distinct functional phenotypes, a process called polarization. Because macrophages have been previously shown to degrade the nuclear envelope proteins lamin A/C upon pro-inflammatory polarization, and lamins are considered key determinants of nuclear deformability, we aimed to address the effect of pro-inflammatory stimulation on nuclear mechanics. We present the surprising finding that polarized bone marrow-derived macrophages have less deformable nuclei than unpolarized macrophages, despite their reduced lamin A/C levels. Furthermore, pro-inflammatory macrophages exhibited altered chromatin dynamics relative to unpolarized macrophages, including redistribution of trimethylated histone H3K9 (H3K9me3) from the nuclear periphery to the interior and increased chromatin compaction. Our findings suggest a model in which pro-inflammatory stimulation of macrophages induces chromatin changes that drive nuclear stiffening, and that in these cells, chromatin, rather than the nuclear lamina, is the major driver for resisting nuclear deformation. These findings may have functional relevance for the physiological function of polarized macrophages, as the mechanical properties of the nucleus can influence how these cells adapt and respond to their environments in the context of cell migration or inflammatory disease pathologies.

cell biology↗

bioRxiv2023

Optical manipulation reveals macrophage mediated mesoscale brain mechanical homeostasis in vivo

Tissues are active materials where epithelial turnover, immune surveillance, and remodeling of stromal cells such as macrophages all regulate form and function. Scattering modalities such as Brillouin microscopy (BM) can non-invasively access mechanical signatures at GHz. However, our traditional understanding of tissue material properties is derived mainly from modalities which probe mechanical properties at different frequencies. Thus, reconciling measurements amongst these modalities remains an active area. Here, we compare optical tweezer active microrheology (OT-AMR) and Brillouin microscopy (BM) to longitudinally map brain development in the larval zebrafish. We determine that each measurement is able to detect a mechanical signature linked to functional units of the brain. We demonstrate that the corrected BM-Longitudinal modulus using a density factor correlates well with OT-AMR storage modulus at lower frequencies. We also show that the brain tissue mechanical properties are dependent on both the neuronal architecture and the presence of macrophages. Moreover, the BM technique is able to delineate the contributions to mechanical properties of the macrophage from that due to colony stimulating factor 1 receptor (CSF1R) mediated stromal remodeling. Here, our data suggest that macrophage remodeling is instrumental in the maintenance of tissue mechanical homeostasis during development. Moreover, the strong agreement between the OT-AM and BM further demonstrates that scattering-based technique is sensitive to both large and minute structural modification in vivo.

biophysics↗

bioRxiv2023

Aberrant tissue stiffness impairs neural tube development in Mthfd1l mutant mouse embryos

Neurulation is a highly synchronized biomechanical process leading to the formation of the brain and spinal cord, and its failure leads to neural tube defects (NTDs). Although we are rapidly learning the genetic mechanisms underlying NTDs, the biomechanical aspects are largely unknown. To understand the correlation between NTDs and tissue stiffness during neural tube closure (NTC), we imaged an NTD murine model using optical coherence tomography (OCT), Brillouin microscopy, and confocal fluorescence microscopy. Here, we associate structural information from OCT with local stiffness from the Brillouin signal of embryos undergoing neurulation. The stiffness of neuroepithelial tissues in Mthfd1l null embryos was significantly lower compared to that of wild-type embryos, while exogenous formate supplementation improved tissue stiffness and gross embryonic morphology in both nullizygous and heterozygous embryos. Our results demonstrate the significance of proper tissue stiffness for normal NTC and pave the way for future studies on the mechanobiology of normal and abnormal embryonic development.

developmental biology↗

bioRxiv2023

Disruption of Fuz in mouse embryos generates hypoplastic hindbrain development and reduced cranial nerve ganglia.

The formation of the brain and spinal cord is initiated in the earliest stages of mammalian pregnancy in a highly organized process known as neurulation. Convergent and extension movements transforms a flat sheet of ectodermal cells into a narrow and elongated line of neuroepithelia, while a major source of Sonic Hedgehog signaling from the notochord induces the overlying neuroepithelial cells to form two apposed neural folds. Afterward, neural tube closure occurs by synchronized coordination of the surface ectoderm and adjacent neuroepithelial walls at specific axial regions known as neuropores. Environmental or genetic interferences can impair neurulation resulting in neural tube defects. The Fuz gene encodes a subunit of the CPLANE complex, which is a macromolecular planar polarity effector required for ciliogenesis. Ablation of Fuz in mouse embryos results in exencephaly and spina bifida, including dysmorphic craniofacial structures due to defective cilia formation and impaired Sonic Hedgehog signaling. In this work, we demonstrate that knocking Fuz out during embryonic mouse development results in a hypoplastic hindbrain phenotype, displaying abnormal rhombomeres with reduced length and width. This phenotype is associated with persistent loss of ventral neuroepithelial stiffness, in a notochord adjacent area at the level of the rhombomere 5, preceding the development of exencephaly in Fuz ablated mutants. The formation of cranial and paravertebral ganglia is also impaired in these embryos, indicating that Fuz has a critical function sustaining normal neural tube development and neuronal differentiation. O_FIG O_LINKSMALLFIG WIDTH=153 HEIGHT=200 SRC="FIGDIR/small/552068v1_ufig1.gif" ALT="Figure 1"> View larger version (77K): org.highwire.dtl.DTLVardef@1c1d63forg.highwire.dtl.DTLVardef@d8e5e0org.highwire.dtl.DTLVardef@1dbf248org.highwire.dtl.DTLVardef@ff6969_HPS_FORMAT_FIGEXP M_FIG C_FIG SIGNIFICANCE STATEMENTNeural tube defects (NTDs) are a common cause of disability in children, representing the second most common congenital structural malformation in humans following only congenital cardiovascular malformations. NTDs affect approximately 1 to 2 pregnancies per 1000 births every year worldwide, when the mechanical forces folding the neural plate fails to close at specific neuropores located anteriorly (cranial) or posteriorly (caudal) along the neural tube, in a process known as neurulation, which happens throughout the third and fourth weeks of human pregnancy.

developmental biology↗

bioRxiv2022

Time-lapse mechanical imaging of neural tube closure in live embryo using Brillouin microscopy

Neural tube closure (NTC) is a complex process of embryonic development involving molecular, cellular, and biomechanical mechanisms. While the genetic factors and biochemical signaling have been extensively investigated, the role of tissue biomechanics remains mostly unexplored due to the lack of tools. Here, we developed a new optical modality that can conduct time-lapse mechanical imaging of neural plate tissue as the embryo is experiencing neurulation. This technique is based on the combination of a confocal Brillouin microscope and an on-stage incubator for the modified ex ovo culturing of chick embryo. With this technique, for the first time, we captured the mechanical evolution of the neural plate tissue with live embryos. Specifically, we observed the continuous stiffening of the neural plate during NTC for ex ovo cultured embryos, which is consistent with the data of in ovo culture as well as previous studies. Beyond that, we found the tissue stiffening was highly correlated with the tissue thickening and bending. We foresee this non-contact and label-free technique can open new opportunities to understand the biomechanical mechanisms in development. Summary statementAn all-optical technique is developed to capture the evolution of tissue mechanics during neural tube closure of live chick embryo.

biophysics↗

bioRxiv2022

Rapid biomechanical imaging at low irradiation level via dual line-scanning Brillouin microscopy

Brillouin microscopy is a promising all-optical technique for biomechanics but is limited by slow acquisition speed and/or large irradiation doses. Here, we introduce multiplexed Brillouin microscopy that overcomes both these limits by over one order of magnitude with selective illumination and single-shot analysis of hundreds of points along the incident beam axis. We demonstrate the enabling capabilities of this method probing rapid response to perturbations and long-term mechanical evolution of tumor spheroids.

bioengineering↗

bioRxiv2022

Multimodal microscale mechanical mapping of cancer cells in complex microenvironments

The mechanical phenotype of the cell is critical for survival following deformations due to confinement and fluid flow. One idea is that cancer cells are plastic and adopt different mechanical phenotypes under different geometries that aid in their survival. Thus, an attractive goal, is to disrupt the cancer cells ability to adopt multiple mechanical states. To begin to address this question, we aimed to quantify the diversity of these mechanical states using in vitro biomimetics to mimic in vivo 2D and 3D extracellular matrix environments. Here, we used two modalities Brillouin microscopy ([~]GHz) and broadband frequency (3-15kHz) optical tweezer microrheology to measure microscale cell mechanics. We measured the response of intracellular mechanics of cancer cells cultured in 2D and 3D environments where we modified substrate stiffness, dimensionality (2D versus 3D), and presence of fibrillar topography. We determined that there was good agreement between two modalities despite the difference in timescale of the two measurements. These findings on cell mechanical phenotype in different environments confirm a correlation between modalities that employ different mechanisms at different temporal scales (Hz-kHz vs. GHz). We also determined that observed heterogeneity in cell shape that is more closely linked to the cells mechanical state. We also determined that individual cells in multicellular spheroids exhibit a lower degree of mechanical heterogeneity when compared to single cells cultured in monodisperse 3D cultures. Moreover, the observed decreased heterogeneity among cells in spheroids suggested that there is mechanical cooperativity between cells that make up a single spheroid.

biophysics↗

bioRxiv2021

Directing multicellular organization by varying the aspect ratio of soft hydrogel microwells

Multicellular organization with precise spatial definition is an essential step in a wide range of biological processes, including morphogenesis, development, and healing. Gradients and patterns of chemoattractants are well-described guides of multicellular organization, but the influences of three-dimensional geometry of soft hydrogels on multicellular organization are less well defined. Here, we report the discovery of a new mode of self-organization of endothelial cells in ring-like patterns on the perimeters of hydrogel microwells that is independent of protein or chemical patterning and is driven only by geometry and substrate stiffness. We observe quantitatively striking influences of both the microwell aspect ratio ({varepsilon} = perimeter/depth) and the hydrogel modulus. We systematically investigate the physical factors of cells and substrates that drive this multicellular behavior and present a mathematical model that explains the multicellular organization based upon balancing extracellular and cytoskeletal forces. These forces are determined in part by substrate stiffness, geometry, and cell density. The force balance model predicts the direction and distance of translational cell migration based on the dynamic interaction between tangential cytoskeletal tension and cell-cell and cell-substrate adhesion. We further show that the experimental observations can be leveraged to drive customized multicellular self-organization. Our observation of this multicellular behavior demonstrates the importance of the combinatorial effects of geometry and stiffness in complex biological processes. It also provides a new methodology for direction of cell organization that may facilitate the engineering of bionics and integrated model organoid systems.

bioengineering↗

bioRxiv2019

Long-term Brillouin imaging of live cells with reduced absorption-mediated damage at 660nm wavelength

In Brillouin microscopy, absorption-induced photodamage of incident light is the primary limitation on signal-to-noise ratio in many practical scenarios. Here we show that 660 nm may represent an optimal wavelength for Brillouin microscopy as it offers minimal absorption-mediated photodamage at high Brillouin scattering efficiency and the possibility to use a pure and narrow laser line from solid-state lasing medium. We demonstrate that live cells are ~80 times less susceptible to the 660 nm incident light compared to 532 nm light, which overall allows Brillouin imaging of up to more than 30 times higher SNR. We show that this improvement enables Brillouin imaging of live biological samples with improved accuracy, higher speed and/or larger fields of views with denser sampling.

biophysics↗

bioRxiv2017

Long-Term Imaging of Cellular Forces with High Precision by Elastic Resonator Interference Stress Microscopy

Cellular forces are crucial for many biological processes but current methods to image them have limitations with respect to online analysis, resolution and throughput. Here, we present a robust approach to measure mechanical cell-substrate interactions in diverse biological systems by interferometrically detecting deformations of an elastic micro-cavity. Elastic Resonator Interference Stress Microscopy (ERISM) yields stress maps with exceptional precision and large dynamic range (2 nm displacement resolution over a >1 m range, translating into 1 pN force sensitivity). This enables investigation of minute vertical stresses (<1 Pa) involved in podosome protrusion, protein specific cell-substrate interaction and amoeboid migration through spatial confinement in real time. ERISM requires no zero-force reference and avoids phototoxic effects, which facilitates force monitoring over multiple days and at high frame rates and eliminates the need to detach cells after measurements. This allows observation of slow processes like differentiation and further investigation of cells, e.g. by immunostaining.

biophysics↗