Biology subjects

Hansen, J. N.

Publications and source records attributed to Hansen, J. N..

24 records · Page 2Linked to original sources

bioRxiv2020

CiliaQ - a simple, open-source software for automated quantification of ciliary morphology and fluorescence in 2D, 3D, and 4D images

Cilia are hair-like membrane protrusions that emanate from the surface of most vertebrate cells and are classified into motile and primary cilia. Motile cilia move fluid flow or propel cells, while also fulfilling sensory functions. Primary cilia are immotile and act as a cellular antenna, translating environmental cues into cellular responses. Ciliary dysfunction leads to severe diseases, commonly termed ciliopathies. The molecular details underlying ciliopathies and ciliary function are, however, not well understood. Since cilia are small subcellular compartments, imaging-based approaches have been used to study them. However, tools to comprehensively analyze images are lacking. Automatic analysis approaches require commercial software and are limited to 2D analysis and only a few parameters. The widely used manual analysis approaches are time consuming, user-biased, and difficult to compare. Here, we present CiliaQ, a package of open-source, freely-available, and easy-to-use ImageJ plugins. CiliaQ allows high throughput analysis of 2D and 3D, static or time-lapse images from fluorescence microscopy of cilia in cell culture or tissues, and outputs a comprehensive list of parameters for ciliary morphology, length, bending, orientation, and fluorescence intensity, making it broadly applicable. We envision CiliaQ as a resource and platform for reproducible and comprehensive analysis of ciliary function in health and disease.

cell biology↗

bioRxiv2020

Translational activity of the splicing factor SRSF1 is required for development and cilia function

Shuttling RNA-binding proteins coordinate nuclear and cytoplasmic steps of gene expression. The SR family proteins regulate RNA splicing in the nucleus and a subset of them, including SRSF1, shuttles between the nucleus and cytoplasm affecting post-splicing processes. However, the physiological significance of this remains unclear. Here, we used genome editing to knock-in a nuclear retention signal (NRS) in Srsf1 to create a mouse model harboring an SRSF1 protein that is retained exclusively in the nucleus. Srsf1NRS/NRS mutants displayed small body size, hydrocephalus and immotile sperm, all traits associated with ciliary defects. We observed reduced translation of a subset of mRNAs and decreased abundance of proteins involved in multiciliogenesis, with disruption of ciliary ultrastructure and motility in cells derived from this mouse model. These results demonstrate that SRSF1 shuttling is used to reprogram gene expression networks in the context of high cellular demands, as observed here, during motile ciliogenesis.

molecular biology↗

bioRxiv2020

Targeting Vitamin-D receptor (VDR) by a small molecule antagonist MeTC7 inhibits PD-L1 but controls THMYCN neuroblastoma growth PD-L1 independently

Vitamin-D receptor (VDR) mRNA is enriched in malignant lung, ovarian and pancreatic tissues and showed poor prognoses. Calcitriol and stable or CRISPR-directed VDR upregulation increased PD-L1mRNA and protein expression in cancer cells in-vitro. A ChIP assay showed the binding of VDR with VDREPD-L1. Stattic, a STAT3 phosphorylation inhibitor blocked calcitriol or VDR overexpression induced PD-L1 upregulation. MeTC7, a VDR antagonist developed by us, reduced PD-L1 expression on macrophages, ovarian, lung, breast, and pancreatic cancer cells in-vitro. In radiotherapy inducible PD-L1 model of orthotopic MC38 murine colon cancer, MeTC7 decreased PD-L1 surface expression, suppressed inflammatory monocytes (IMs) population and increased intra-tumoral CD69+PD1+CD8+T-cells. Intriguingly, MeTC7 reduced TH-MYCN transgenic neuroblastoma tumor growth without affecting PD-L1 and tumor immune milieu. In summary, Vitamin-D/VDR drives PD-L1 expression on cancer cells via STAT-3. Inhibiting VDR exhibited anti-checkpoint effects in orthotopic colon tumors, whereas PDL1-independent and anti-VDR/MYCN effects controlled growth of transgenic neuroblastoma and xenografted tumors. SummaryVitamin-D/VDR induces PD-L1 expression on cancer cells via STAT-3; and targeting VDR by a novel small molecule antagonist MeTC7 exhibits both anti-PD-L1 and anti-VDR/MYCN effects in tumor models.

cancer biology↗

bioRxiv2020

Multifocal imaging for precise, label-free tracking of fast biological processes in 3D

Many biological processes happen on a nano-to millimeter scale and within milliseconds. Established methods such as confocal microscopy are suitable for precise 3D recordings but lack the temporal or spatial resolution to resolve fast 3D processes and require labeled samples. Multifocal imaging (MFI) allows high-speed 3D imaging but is limited by the compromise between high spatial resolution and large field-of-view (FOV), and the requirement for bright fluorescent labels. Here, we provide a new open-source 3D reconstruction algorithm for multifocal images that allows using MFI for fast, precise, label-free tracking spherical and filamentous structures in a large FOV and across a high depth. We characterize fluid flow and flagellar beating of human and sea urchin sperm with a high z-precision of 0.15 m, in a large volume of 240 x 260 x 21 m, and at high speed (500 Hz). The large sampling volume allowed to follow sperm trajectories while simultaneously recording their flagellar beat. Our MFI concept is cost-effective, can be easily implemented, and does not rely on object labeling, which renders it broadly applicable.

cell biology↗

bioRxiv2020

Nanobody-directed targeting of optogenetic tools reveals differential regulation of cilia length

Compartmentalization of cellular signaling forms the molecular basis of cellular behavior. The primary cilium constitutes a subcellular compartment that orchestrates signal transduction independent from the cell body. Ciliary dysfunction causes severe diseases, termed ciliopathies. Analyzing ciliary signaling and function has been challenging due to the lack of tools to temporarily manipulate and analyze ciliary signaling. Here, we describe a nanobodybased targeting approach for optogenetic tools that is applicable in vitro and in vivo and allows to specifically analyze ciliary signaling and function. Thereby, we overcome the loss of protein function observed after direct fusion to a ciliary targeting sequence. We functionally localized modifiers of cAMP signaling, i.e. the photo-activated adenylate cyclase bPAC and the light-activated phosphodiesterase LAPD, as well as the cAMP biosensor mlCNBD-FRET to the cilium. Using this approach, we studied the contribution of spatial cAMP signaling in controlling cilia length. Combining optogenetics with nanobody-based targeting will pave the way to the molecular understanding of ciliary function in health and disease.

cell biology↗

bioRxiv2018

SpermQ - a simple analysis software to comprehensively study flagellar beating and sperm steering

Motile cilia, also called flagella, drive cell motility across a broad range of species; some cilia propel prokaryotes and eukaryotic cells like sperm, while cilia on epithelial surfaces create complex fluid patterns e.g. in the brain or lung. For sperm, the picture has emerged that the motile cilium, also called flagellum, is not only a motor, but also a sensor that detects stimuli from the environment, computing the beat pattern according to the sensory input. Thereby, the flagellum of the sperm cell navigates the sperm through a complex environment like the female genital tract. However, we know very little about how environmental signals change the flagellar beat and, thereby, the swimming behaviour of sperm. It has been proposed that distinct signalling domains in the flagellum control the flagellar beat. A detailed analysis has been mainly hampered by the fact that current comprehensive analysis approaches rely on complex microscopy and analysis systems. Thus, knowledge on sperm signalling regulating the flagellar beat is based on custom quantification approaches that are limited to only a few aspects of the flagellar beat, do not resolve the kinetics of the entire flagellum, rely on manual, qualitative descriptions, and are little comparable among each other. Here, we present SpermQ, a ready-to-use and comprehensive analysis software to quantify sperm motility. SpermQ provides a detailed quantification of the flagellar beat based on common time-lapse images acquired by dark-field or epi-fluorescence microscopy, making SpermQ widely applicable. We envision SpermQ becoming a standard tool in flagellar and motile cilia research that allows to readily link studies on individual signalling components in sperm and distinct flagellar beat patterns.

cell biology↗