Anusha B. Gopalan

European Molecular Biology Laboratory

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Alba Diz-Muñoz

European Molecular Biology Laboratory

Simone Köhler

European Molecular Biology Laboratory

Maria Bernabeu

European Molecular Biology Laboratory

Georgia Rapti

Heidelberg University

Richard R. Sprenger

University of Southern Denmark

Robert Prevedel

European Molecular Biology Laboratory

Jan Ellenberg

European Molecular Biology Laboratory

Christer S. Ejsing

University of Southern Denmark

Matt Govendir

European Molecular Biology Laboratory

Carlo Bevilacqua

European Molecular Biology Laboratory

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European Molecular Biology Laboratory

Heidelberg University

European Molecular Biology Laboratory

University of Southern Denmark

Harvard University

University of Toronto

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European Molecular Biology Organization

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Caging of membrane-to-cortex attachment proteins can trigger cellular symmetry breaking

bioRxiv (Cold Spring Harbor Laboratory) (2024)

Srishti Dar Rubén Moreno Ivan Palaia Anusha B. Gopalan Zachary Gao Sun

SUMMARY To migrate, divide, and change shape, cells must regulate the mechanics of their periphery. The cell surface is a complex structure that consists of a thin, contractile cortical actin network tethered to the plasma membrane by specialized membrane-to-cortex attachment (MCA) proteins. This active and constantly fluctuating system maintains a delicate mechanochemical state which permits spontaneous polarization and shape change when needed. Combining in silico , in vitro , and in vivo experiments we show how membrane viscosity and MCA protein length regulate cortical dynamics. We reveal a novel mechanism whereby caging of linker proteins in the actin cortex allows for the amplification of small changes in these key parameters, leading to major alterations of cortical contractility. In cells, this mechanism alone gives rise to symmetry breaking phenomena, suggesting that local changes in lipid composition, in combination with the choice of MCA proteins, contribute to the regulation of cellular morphogenesis and function.

Cell Cortex

Contractility

Cell membrane

10.1101/2024.10.14.618153

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Lipotype acquisition during neural development is not recapitulated in stem cell–derived neurons

Life Science Alliance (2024)

Anusha B. Gopalan Lisa van Uden Richard R. Sprenger Nadine Fernandez-Novel Marx Helle Bogetofte

During development, different tissues acquire distinct lipotypes that are coupled to tissue function and homeostasis. In the brain, where complex membrane trafficking systems are required for neural function, specific glycerophospholipids, sphingolipids, and cholesterol are highly abundant, and defective lipid metabolism is associated with abnormal neural development and neurodegenerative disease. Notably, the production of specific lipotypes requires appropriate programming of the underlying lipid metabolic machinery during development, but when and how this occurs is unclear. To address this, we used high-resolution MS ALL lipidomics to generate an extensive time-resolved resource of mouse brain development covering early embryonic and postnatal stages. This revealed a distinct bifurcation in the establishment of the neural lipotype, whereby the canonical lipid biomarkers 22:6-glycerophospholipids and 18:0-sphingolipids begin to be produced in utero, whereas cholesterol attains its characteristic high levels after birth. Using the resource as a reference, we next examined to which extent this can be recapitulated by commonly used protocols for in vitro neuronal differentiation of stem cells. Here, we found that the programming of the lipid metabolic machinery is incomplete and that stem cell–derived cells can only partially acquire a neural lipotype when the cell culture media is supplemented with brain-specific lipid precursors. Altogether, our work provides an extensive lipidomic resource for early mouse brain development and highlights a potential caveat when using stem cell–derived neuronal progenitors for mechanistic studies of lipid biochemistry, membrane biology and biophysics, which nonetheless can be mitigated by further optimizing in vitro differentiation protocols.

Glycerophospholipids

Neural Development

Sphingolipid

10.26508/lsa.202402622

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Citations (4)

Author Reply to Peer Reviews of Lipotype acquisition during neural development in vivo is not recapitulated in stem cell-derived neurons

Anusha B. Gopalan Lisa van Uden Richard R. Sprenger Nadine Fernandez-Novel Marx Helle Bogetofte

Neural Development

10.15252/rc.2022337090

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Author Correction: Pulsed stimulated Brillouin microscopy enables high-sensitivity mechanical imaging of live and fragile biological specimens

Nature Methods (2024)

Fan Yang Carlo Bevilacqua Sebastian Hambura Ana Rita Rodrigues Neves Anusha B. Gopalan

In the version of the article initially published, in Fig. 3c, d andg, the central canal was mislabeled as a blood vessel.In the fourth paragraph of the introduction, first and third paragraphs of the "High-specificity SBS imaging in live zebrafish and C. elegans" section and the Fig. 3 legend, mentions of blood vessels have been amended to discuss the spinal chord or central canal regions.Additionally, reference 28 has been changed to Thouvenin, O. et al.Origin and role of the cerebrospinal fluid bidirectional flow in the central canal.

Brillouin zone

10.1038/s41592-024-02259-w

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Pulsed stimulated Brillouin microscopy enables high-sensitivity mechanical imaging of live and fragile biological specimens

Zenodo (CERN European Organization for Nuclear Research) (2023)

Fan Yang Carlo Bevilacqua Sebastian Hambura Ana Rita Rodrigues Neves Anusha B. Gopalan

Brillouin zone

Live cell imaging

10.5281/zenodo.8211867

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Pulsed stimulated Brillouin microscopy enables high-sensitivity mechanical imaging of live and fragile biological specimens

Nature Methods (2023)

Fan Yang Carlo Bevilacqua Sebastian Hambura Ana Rita Rodrigues Neves Anusha B. Gopalan

Abstract Brillouin microscopy is an emerging optical elastography technique capable of assessing mechanical properties of biological samples in a three-dimensional, all-optical and noncontact fashion. The typically weak Brillouin scattering signal can be substantially enhanced via a stimulated Brillouin scattering (SBS) process; however, current implementations require high pump powers, which prohibit applications to photosensitive or live imaging of biological samples. Here we present a pulsed SBS scheme that takes advantage of the nonlinearity of the pump–probe interaction. In particular, we show that the required pump laser power can be decreased ~20-fold without affecting the signal levels or spectral precision. We demonstrate the low phototoxicity and high specificity of our pulsed SBS approach by imaging, with subcellular detail, sensitive single cells, zebrafish larvae, mouse embryos and adult Caenorhabditis elegans . Furthermore, our method permits observing the mechanics of organoids and C. elegans embryos over time, opening up further possibilities for the field of mechanobiology.

Brillouin zone

Live cell imaging

SIGNAL (programming language)

mechanobiology

Biological specimen

10.1038/s41592-023-02054-z

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Citations (23)

Pulsed stimulated Brillouin microscopy enables high-sensitivity mechanical imaging of live and fragile biological specimens

bioRxiv (Cold Spring Harbor Laboratory) (2022)

Fan Yang Carlo Bevilacqua Sebastian Hambura Ana Rita Rodrigues Neves Anusha B. Gopalan

Brillouin microscopy is an emerging optical elastography technique capable of assessing mechanical properties of biological samples in a 3D, all-optical and hence non-contact fashion. The typically weak Brillouin scattering signal can be substantially enhanced via a stimulated photon-phonon process, which improves the signal-to-background ratio (SBR) as well as provides higher spectral resolution. However, current implementations of stimulated Brillouin spectroscopy (SBS) require high pump powers, which prohibit applications in many areas of biology, especially when studying photosensitive samples, or when live-imaging in 3D and/or over extended time periods. Here, we present a pulsed SBS scheme that takes full advantage of the non-linearity of the pump-probe interaction in SBS. In particular, we show that through quasi-pulsing and diligent optimization of signal detection parameters, the required pump laser power can be decreased ~20-fold without affecting the signal levels or spectral precision. Moreover, we devise a custom analysis approach that facilitates the analysis of complex, multi-peaked Brillouin spectra in order to harness the high spectral resolution of SBS for the specific identification of biomechanical components inside the point-spread function of the microscope. We then demonstrate the low-phototoxicity and high-specificity of our pulsed SBS approach by imaging sensitive single cells, zebrafish larvae, and mouse embryos as well as adult C. elegans with sub-cellular detail. Furthermore, our method permits observing the mechanics of organoids and C. elegans embryos over time. We expect that the substantially lower photo-burden and improved SBR of pulsed SBS will facilitate studying biomechanics in 3D at high spatio-temporal resolution in living biological specimens in a non-invasive manner, opening up exciting new possibilities for the field of mechanobiology.

Brillouin zone

SIGNAL (programming language)

Live cell imaging

10.1101/2022.11.10.515835

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Citations (3)

Lipotype acquisition during neural developmentin vivois not recapitulated in stem cell-derived neurons

bioRxiv (Cold Spring Harbor Laboratory) (2022)

Anusha B. Gopalan Lisa van Uden Richard R. Sprenger Nadine Fernandez-Novel Marx Helle Bogetofte

Abstract During development, different tissues acquire distinct lipotypes that are coupled to tissue function and homeostasis. In the brain, where complex membrane trafficking systems are required for neural function, specific glycerophospholipids, sphingolipids, and cholesterol are highly abundant, and defective lipid metabolism is associated with abnormal neural development and neurodegenerative disease. Notably, the production of tissue-specific lipotypes requires appropriate programming of the underlying lipid metabolic machinery, but when and how this occurs is unclear. To address this, we used high-resolution mass spectrometry-based (MS ALL ) lipidomics to perform a quantitative and comprehensive analysis of mouse brain development covering early embryonic and postnatal stages. We discovered a distinct bifurcation in the establishment of the neural lipotype, whereby the canonical brain lipid biomarkers 22:6-glycerophospholipids and 18:0-sphingolipids begin to be produced in utero , whereas cholesterol attains its characteristic high levels after birth. In contrast, when profiling rodent and human stem cell-derived neurons, we observed that these do not acquire a brain lipotype per se . However, upon probing the lipid metabolic wiring by supplementing brain lipid precursors, we found that the stem cell-derived neurons were partially able to establish a brain-like lipotype, demonstrating that the cells are partially metabolically committed. Altogether, our report provides an extensive lipidomic resource for brain development and highlights a potential challenge in using stem cell-derived neurons for mechanistic studies of lipid biochemistry, membrane biology and biophysics that can be mitigated by further optimizing in vitro differentiation protocols. Significance Statement We report an extensive time-resolved resource of lipid molecule abundances across mouse brain development, starting as early as 10 days post-fertilization. The resource reveals a bifurcation in the establishment of the neural lipotype where the canonical 22:6-glycerophospholipid and 18:0-sphingolipid biomarkers are attained in utero , whereas cholesterol is attained after birth. Furthermore, we uncover that the neural lipotype is not established in rodent and human stem cell-derived neurons in vitro .

Glycerophospholipids

Sphingolipid

Neural Development

10.1101/2022.08.31.505694

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Citations (2)

CPMG Experiments for Protein Minor Conformer Structure Determination

Methods in molecular biology (2017)

Anusha B. Gopalan D. Flemming Hansen Pramodh Vallurupalli

Minor (academic)

Conformational isomerism

10.1007/978-1-4939-7386-6_11