Strong intracellular signal inactivation produces sharper and more robust signaling from cell membrane to nucleus

For a chemical signal to propagate across a cell, it must navigate a tortuous environment involving a variety of organelle barriers. In this work we study mathematical models for a basic chemical signal, the arrival times at the nuclear membrane of proteins that are activated at the cell membrane and diffuse throughout the cytosol. Organelle surfaces within human B cells are reconstructed from soft X-ray tomographic images, and modeled as reflecting barriers to the molecules’ diffusion. We show that signal inactivation sharpens signals, reducing variability in the arrival time at the nuclear membrane. Inactivation can also compensate for an observed slowdown in signal propagation induced by the presence of organelle barriers, leading to arrival times at the nuclear membrane that are comparable to models in which the cytosol is treated as an open, empty region. In the limit of strong signal inactivation this is achieved by filtering out molecules that traverse non-geodesic paths.https://www.biorxiv.org/content/10.1101/2020.01.16.909333v1First author draf

Quantitatively Imaging Chromosomes by Correlated Cryo-Fluorescence and Soft X-Ray Tomographies

AbstractSoft x-ray tomography (SXT) is increasingly being recognized as a valuable method for visualizing and quantifying the ultrastructure of cryopreserved cells. Here, we describe the combination of SXT with cryogenic confocal fluorescence tomography (CFT). This correlative approach allows the incorporation of molecular localization data, with isotropic precision, into high-resolution three-dimensional (3-D) SXT reconstructions of the cell. CFT data are acquired first using a cryogenically adapted confocal light microscope in which the specimen is coupled to a high numerical aperture objective lens by an immersion fluid. The specimen is then cryo-transferred to a soft x-ray microscope (SXM) for SXT data acquisition. Fiducial markers visible in both types of data act as common landmarks, enabling accurate coalignment of the two complementary tomographic reconstructions. We used this method to identify the inactive X chromosome (Xi) in female v-abl transformed thymic lymphoma cells by localizing enhanced green fluorescent protein-labeled macroH2A with CFT. The molecular localization data were used to guide segmentation of Xi in the SXT reconstructions, allowing characterization of the Xi topological arrangement in near-native state cells. Xi was seen to adopt a number of different topologies with no particular arrangement being dominant

Selective targeting of WNT receptor FZD7 in human pluripotent stem cells and ovarian cancer

WNT signaling is a highly conserved pathway with dual roles in development and disease. In human pluripotent stem (hPS) cells, an in vitro model of mammalian development, low-level WNT signaling maintains the self-renewal state, but carefully timed increases in WNT can induce terminal differentiation. While the WNT receptor Frizzled-7 (FZD7) is an established regulator of pluripotency in hPS cells, it is unclear if FZD7 or additional FZD receptors mediate the WNT signal that promotes stem cell differentiation. To address whether FZD7 is required for this process, my co-authors and I engineered bispecific antibody F7L6, which acts as a WNT signaling agonist by selectively heterodimerizing FZD7 and its coreceptor, LRP6. F7L6 treatment of hPS cells elicited a transcriptional response similar to that observed for Wnt3a treatment, thus establishing that FZD7 signaling is sufficient to promote mesendodermal differentiation. Though WNT signaling is required for normal embryonic development, it can be dysregulated in diseases such as cancer. Targeting WNT signaling in solid tumors has been historically challenging due to the WNT requirement for bone homeostasis. Clinical pan-WNT inhibitors frequently induce bone-related adverse events, creating a need for more specific WNT-pathway targeting strategies. We identified elevated RNA expression of FZD7 in aggressive subtypes of ovarian serous cystadenocarcinoma (OV) in The Cancer Genome Atlas and confirmed high FZD7 protein expression in OV, but low FZD7 in normal ovary tissues, indicating that FZD7 is a tumor-specific antigen. We developed novel antibody-drug conjugate (F7-ADC), septuximab vedotin, a chimeric human-mouse IgG1 antibody to human FZD7 conjugated to antimitotic payload drug MMAE. The F7-ADC specifically binds FZD7, potently kills ovarian cancer cells in vitro, and induces regression of ovarian tumor xenografts in nude mouse models. To evaluate F7-ADC toxicity in vivo, we engineered immunocompetent Fzd7hF7/hF7 mice to express Fzd7P188L receptors reactive with our human-targeting F7-ADC. F7-ADC treatment did not induce adverse effects on Fzd7hF7/hF7 mouse health or histopathological changes at the tissue level. Overall, our data suggest that the F7-ADC approach may be a powerful strategy to combat FZD7-expressing ovarian cancers in the clinic

Imaging and characterizing cells using tomography

We can learn much about cell function by imaging and quantifying sub-cellular structures, especially if this is done non-destructively without altering said structures. Soft X-ray tomography (SXT) is a high-resolution imaging technique for visualizing cells and their interior structure in 3D. A tomogram of the cell, reconstructed from a series of 2D projection images, can be easily segmented and analyzed. SXT has a very high specimen throughput compared to other high-resolution structure imaging modalities; for example, tomographic data for reconstructing an entire eukaryotic cell is acquired in a matter of minutes. SXT visualizes cells without the need for chemical fixation, dehydration, or staining of the specimen. As a result, the SXT reconstructions are close representations of cells in their native state. SXT is applicable to most cell types. The deep penetration of soft X-rays allows cells, even mammalian cells, to be imaged without being sectioned. Image contrast in SXT is generated by the differential attenuation soft X-ray illumination as it passes through the specimen. Accordingly, each voxel in the tomographic reconstruction has a measured linear absorption coefficient (LAC) value. LAC values are quantitative and give rise to each sub-cellular component having a characteristic LAC profile, allowing organelles to be identified and segmented from the milieu of other cell contents. In this chapter, we describe the fundamentals of SXT imaging and how this technique can answer real world questions in the study of the nucleus. We also describe the development of correlative methods for the localization of specific molecules in a SXT reconstruction. The combination of fluorescence and SXT data acquired from the same specimen produces composite 3D images, rich with detailed information on the inner workings of cells

Correlative cryogenic tomography of cells using light and soft x-rays

Correlated imaging is the process of imaging a specimen with two complementary modalities, and then combining the two data sets to create a highly informative, composite view. A recent implementation of this concept has been the combination of soft x-ray tomography (SXT) with fluorescence cryogenic microscopy (FCM). SXT-FCM is used to visualize cells that are held in a near-native, cryopreserved. The resultant images are, therefore, highly representative of both the cellular architecture and molecular organization in vivo. SXT quantitatively visualizes the cell and sub-cellular structures; FCM images the spatial distribution of fluorescently labeled molecules. Here, we review the characteristics of SXT-FCM, and briefly discuss how this method compares with existing correlative imaging techniques. We also describe how the incorporation of a cryo-rotation stage into a cryogenic fluorescence microscope allows acquisition of fluorescence cryogenic tomography (FCT) data. FCT is optimally suited for correlation with SXT, since both techniques image the specimen in 3-D, potentially with similar, isotropic spatial resolution

Quantitatively imaging chromosomes by correlated cryo-fluorescence and soft x-ray tomographies.

Soft x-ray tomography (SXT) is increasingly being recognized as a valuable method for visualizing and quantifying the ultrastructure of cryopreserved cells. Here, we describe the combination of SXT with cryogenic confocal fluorescence tomography (CFT). This correlative approach allows the incorporation of molecular localization data, with isotropic precision, into high-resolution three-dimensional (3-D) SXT reconstructions of the cell. CFT data are acquired first using a cryogenically adapted confocal light microscope in which the specimen is coupled to a high numerical aperture objective lens by an immersion fluid. The specimen is then cryo-transferred to a soft x-ray microscope (SXM) for SXT data acquisition. Fiducial markers visible in both types of data act as common landmarks, enabling accurate coalignment of the two complementary tomographic reconstructions. We used this method to identify the inactive X chromosome (Xi) in female v-abl transformed thymic lymphoma cells by localizing enhanced green fluorescent protein-labeled macroH2A with CFT. The molecular localization data were used to guide segmentation of Xi in the SXT reconstructions, allowing characterization of the Xi topological arrangement in near-native state cells. Xi was seen to adopt a number of different topologies with no particular arrangement being dominant

Strong intracellular signal inactivation produces sharper and more robust signaling from cell membrane to nucleus.

For a chemical signal to propagate across a cell, it must navigate a tortuous environment involving a variety of organelle barriers. In this work we study mathematical models for a basic chemical signal, the arrival times at the nuclear membrane of proteins that are activated at the cell membrane and diffuse throughout the cytosol. Organelle surfaces within human B cells are reconstructed from soft X-ray tomographic images, and modeled as reflecting barriers to the molecules' diffusion. We show that signal inactivation sharpens signals, reducing variability in the arrival time at the nuclear membrane. Inactivation can also compensate for an observed slowdown in signal propagation induced by the presence of organelle barriers, leading to arrival times at the nuclear membrane that are comparable to models in which the cytosol is treated as an open, empty region. In the limit of strong signal inactivation this is achieved by filtering out molecules that traverse non-geodesic paths

Strong intracellular signal inactivation produces sharper and more robust signaling from cell membrane to nucleus.

For a chemical signal to propagate across a cell, it must navigate a tortuous environment involving a variety of organelle barriers. In this work we study mathematical models for a basic chemical signal, the arrival times at the nuclear membrane of proteins that are activated at the cell membrane and diffuse throughout the cytosol. Organelle surfaces within human B cells are reconstructed from soft X-ray tomographic images, and modeled as reflecting barriers to the molecules' diffusion. We show that signal inactivation sharpens signals, reducing variability in the arrival time at the nuclear membrane. Inactivation can also compensate for an observed slowdown in signal propagation induced by the presence of organelle barriers, leading to arrival times at the nuclear membrane that are comparable to models in which the cytosol is treated as an open, empty region. In the limit of strong signal inactivation this is achieved by filtering out molecules that traverse non-geodesic paths