Biophysical Analysis of Mechanical Signals in Immotile Cilia of Mouse Embryonic Nodes Using Advanced Microscopic Techniques

Abstract

Immotile cilia of crown cells at the node of mouse embryos are required for sensing leftward fluid flow that gives rise to the breaking of left-right (L-R) symmetry. The flow-sensing mechanism has long remained elusive, mainly because of difficulties inherent in manipulating and precisely analyzing the cilium. Recent progress in optical microscopy and biophysical analysis has allowed us to study the mechanical signals involving primary cilia. In this study, we used high-resolution imaging with mechanical modeling to assess the membrane tension in a single cilium. Optical tweezers, a technique used to trap sub-micron-sized particles with a highly focused laser beam, allowed us to manipulate individual cilia. Super-resolution microscopy allowed us to discern the precise localization of ciliary proteins. Using this protocol, we provide a method for applying these techniques to cilia in mouse embryonic nodes. This method is widely applicable to the determination of mechanical signals in other cilia.

BMP4 regulates asymmetric Pkd2 distribution in mouse nodal immotile cilia and ciliary mechanosensing required for left–right determination

“…28 We determined the angular distribution of Pkd2::Venus protein on the transverse plane of the axoneme by measuring the Venus intensity and angle from the center of gravity of acetylated tubulin (Figure 2A). 29 Analysis of the STED images revealed that cilia in BMP4-treated embryos exhibited a significantly biased Pkd2 distribution toward the dorsal side with a D/(D + V) ratio of 0.65 ± 0.14 ( n = 40; Figure 2B, C) compared with that in the control embryos with a ratio of 0.54 ± 0.12, as previously reported. 5 In contrast, cilia on Noggin-treated embryos showed an asymmetric distribution with a D/(D + V) ratio of 0.55 ± 0.09 ( n = 36), which was similar to that in the control (Figure 2B, D).…”

“…5 Briefly, iv/iv embryos harboring the NDE4 – hsp – 5HT 6 – GCaMP6 – 2A – 5HT 6 – mCherry 5,7 and NDE4 – hsp – GCaMP6 5 transgenes were cultured from the LB to 0–2 ss in the presence of recombinant BMP4 or Noggin protein (Figure 4A). The frequency of calcium transients in the cytoplasm was assessed while applying dorsal and ventral bending of the cilium using a 3.5-μm-diameter bead trapped using optical tweezers 5,29,31 (Figure 4A). Immotile cilia in the left–right organizer of mouse 5 and zebrafish 32 embryos function as mechanosensors.…”

“…Pkd2 localization in immotile cilia was analyzed as previously described. 5,29 After roller culture, the embryos were washed with PBS. Immunostaining was performed as previously described.…”

“…Immunostaining was performed as previously described. 5,29 Anti-acetylated tubulin (1:200 dilution; T6793; Sigma-Aldrich) and anti-green fluorescent protein (1:200 dilution; ab13970; Abcam, Cambridge, UK) primary antibodies were used. The pair of secondary antibodies used were anti-chick and anti-mouse conjugated either with AlexaFluor Plus 488 (1:200 dilution; A32931; Invitrogen, Carlsbad, CA, USA) and AlexaFluor Plus 555 (1:200 dilution; A32727; Invitrogen) for Airyscan observation or STAR ORANGE (1:200 dilution; anti-chick; Abberior, Göttingen, Germany) and STAR RED (1:200 dilution; anti-mouse; Abberior) for STED observation.…”

Optical microscopic imaging, manipulation, and analysis methods for morphogenesis research

“…The system can be extended to simultaneous two-color live 3D imaging, making it suitable for applications such as calcium imaging [ 13 ]. Most spinning-disk confocal microscopes are installed at camera ports, enabling the construction of a custom-built optical pathway, such as a pathway for optical stimulation or optical tweezers, using an episcopic illuminator port [ 14 , 15 ].…”