Primary cilia and their effects on immune cell functions and metabolism : a model

Primary cilia are hair-like protrusions of the plasma membrane that function as cellular antennae and are present on most cells in the human body. Primary cilia dysfunction leads to severe diseases, commonly termed ‘ciliopathies’. A significant symptom of certain ciliopathies is obesity, and current research aims to identify contributing mechanisms of obesity development in these patients. Western lifestyle-associated factors can trigger chronic inflammation, or metaflammation, which can also attribute to obesity-associated metabolic disorders. However, obese individuals can also be ‘metabolically healthy’, as discussed for a subset of patients with obesity and ciliopathy. Here, we propose that primary cilia signaling might modulate specific immune cell phenotypes, behaviors, and functions, which might impact inflammatory responses in the context of ciliopathies and beyond

Isolation and Flow Cytometry Analysis of Macrophages from White Adipose Tissue

Macrophages are one of the prominent leukocyte populations in white adipose tissue (WAT) and play an important role during WAT homeostasis and remodeling. Macrophage function in WAT is determined by ontogeny and the local tissue environment. Here, we present a protocol to analyze different macrophage populations from murine WAT using flow cytometry

Author Correction: Sensory Primary Cilium is a Responsive cAMP Microdomain in Renal Epithelia

Correction to: Scientific Reports https://doi.org/10.1038/s41598-019-43002-2, published online 25 April 2019 The legend for Figure 1c is incomplete. ‘Time-lapse images represent the intracellular calcium level in response to fluid-shear stress (arrow) by epithelial and endothelial cells treated without (control, vehicle) and with tolvaptan (0.1 μM). Color bar indicates intracellular calcium level from low (black) to high (red). Corresponding brightfield images are shown in Supplementary Fig. S1.’ should read: ‘Time-lapse images represent the intracellular calcium level in response to fluid-shear stress (arrow) by epithelial and endothelial cells that were first treated with vehicle alone (control), and then treated with tolvaptan (0.1 μM) for 20 hours. Color bar indicates intracellular calcium level from low (black) to high (red). Corresponding brightfield images are shown in Supplementary Fig. S1.

Sensory Primary Cilium is a Responsive cAMP Microdomain in Renal Epithelia

Primary cilia are hair-like cellular extensions that sense microenvironmental signals surrounding cells. The role of adenylyl cyclases in ciliary function has been of interest because the product of adenylyl cyclase activity, cAMP, is relevant to cilia-related diseases. In the present study, we show that vasopressin receptor type-2 (V2R) is localized to cilia in kidney epithelial cells. Pharmacologic inhibition of V2R with tolvaptan increases ciliary length and mechanosensory function. Genetic knockdown of V2R, however, does not have any effect on ciliary length, although the effect of tolvaptan on ciliary length is dampened. Our study reveals that tolvaptan may have a cilia-specific effect independent of V2R or verapamil-sensitive calcium channels. Live-imaging of single cilia shows that V2R activation increases cilioplasmic and cytoplasmic cAMP levels, whereas tolvaptan mediates cAMP changes only in a cilia-specific manner. Furthermore, fluid-shear stress decreases cilioplasmic, but not cytoplasmic cAMP levels. Our data indicate that cilioplasmic and cytoplasmic cAMP levels are differentially modulated. We propose that the cilium is a critical sensor acting as a responsive cAMP microcompartment during physiologically relevant stimuli

Nucleo-cytoplasmic shuttling of 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 and tissues 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

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 an open-source 3D reconstruction algorithm for multi-focal 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 z-precision of 0.15 µm, in a volume of 240 × 260 × 21 µm, and at high speed (500 Hz). The 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

The Enigmatic Role of GBA2 in Controlling Locomotor Function

The non-lysosomal glucosylceramidase GBA2 catalyzes the hydrolysis of glucosylceramide to glucose and ceramide. Loss of GBA2 function results in accumulation of glucosylceramide. Mutations in the human GBA2 gene have been associated with hereditary spastic paraplegia (HSP) and autosomal-recessive cerebellar ataxia (ARCA). Patients suffering from these disorders exhibit impaired locomotion and neurological abnormalities. GBA2 mutations found in these patients have been proposed to impair GBA2 function. However, the molecular mechanism underlying the occurrence of mutations in the GBA2 gene and the development of locomotor dysfunction is not well-understood. In this review, we aim to summarize recent findings regarding mutations in the GBA2 gene and their impact on GBA2 function in health and disease

Figure 3i

Sirius Red and Elastin-van-Giesson staining of gWAT from lean Bbs8+/+ and Bbs8-/- mice. Scale bar = 50 µm.</p

Figure 5b

Fluorescence confocal images of lineage-depleted iWAT SVF from Bbs8+/+ and Bbs8-/- mice, labeled against Smoothened (green, SMO) ARL13B (red, cilia), g-Tubulin (magenta, basal body), and with DAPI (blue). Cells were treated with H2O (control) or 1 µM SAG for 24 h. Scale bars are indicated. In all images, the green channel (SMO) was shifted by 10 px to the bottom for better visualizing SMO accumulation in cilia.</p