Chondrocyte-specific response to stiffness-mediated primary cilia formation and centriole positioning.

Mechanical stress and the stiffness of the extracellular matrix are key drivers of tissue development and homeostasis. Aberrant mechanosensation is associated with a wide range of pathologies, including osteoarthritis. Matrix (or substrate) stiffness plays a major role in cell spreading, adhesion, proliferation, and differentiation. However, how specific cells sense substrate stiffness still remains unclear. The primary cilium is an essential cellular organelle that senses and integrates mechanical and chemical signals from the extracellular environment. We hypothesized that the primary cilium dynamically alters its length and position to fine-tune cell mechanosignaling based on substrate stiffness alone. We used a hydrogel system of varying substrate stiffness to examine the role of stiffness on cilia frequency, length, and centriole position as well as cell and nuclei area over time. Contrary to other cell types, we show that chondrocyte primary cilia shorten on softer substrates, demonstrating tissue-specific mechanosensing that is aligned with the tissue stiffness the cells originate from. We further show that stiffness determines centriole positioning to either the basal or apical membrane during attachment and spreading, with centrioles positioned toward the basal membrane on stiffer substrates. These phenomena are mediated by force generation actin-myosin stress fibers in a time-dependent manner. Finally, we show on stiff substrates that primary cilia are involved in tension-mediated cell spreading. We propose that substrate stiffness plays a role in cilia positioning, regulating cellular responses to external forces, and maybe a key driver of mechanosignaling-associated diseases.

Genetic Admixture in the Culturally Unique Peranakan Chinese Population in Southeast Asia.

The Peranakan Chinese are culturally unique descendants of immigrants from China who settled in the Malay Archipelago ∼300-500 years ago. Today, among large communities in Southeast Asia, the Peranakans have preserved Chinese traditions with strong influence from the local indigenous Malays. Yet, whether or to what extent genetic admixture co-occurred with the cultural mixture has been a topic of ongoing debate. We performed whole-genome sequencing (WGS) on 177 Singapore (SG) Peranakans and analyzed the data jointly with WGS data of Asian and European populations. We estimated that Peranakan Chinese inherited ∼5.62% (95% confidence interval [CI]: 4.76-6.49%) Malay ancestry, much higher than that in SG Chinese (1.08%, 0.65-1.51%), southern Chinese (0.86%, 0.50-1.23%), and northern Chinese (0.25%, 0.18-0.32%). A sex-biased admixture history, in which the Malay ancestry was contributed primarily by females, was supported by X chromosomal variants, and mitochondrial (MT) and Y haplogroups. Finally, we identified an ancient admixture event shared by Peranakan Chinese and SG Chinese ∼1,612 (95% CI: 1,345-1,923) years ago, coinciding with the settlement history of Han Chinese in southern China, apart from the recent admixture event with Malays unique to Peranakan Chinese ∼190 (159-213) years ago. These findings greatly advance our understanding of the dispersal history of Chinese and their interaction with indigenous populations in Southeast Asia.

Visualising Neutrophil Actin Dynamics in Zebrafish in Response to Laser Wounding Using Two-Photon Microscopy.

Cells need to migrate along gradients of chemicals (chemotaxis) in the course of development, wound healing, or immune responses. Neutrophils are prototypical migratory cells that are rapidly recruited to injured or infected tissues from the bloodstream. Their chemotaxis to these inflammatory sites involves changes in cytoskeletal dynamics in response to gradients of chemicals produced therein. Neutrophil chemotaxis has been largely studied in vitro; few assays have been developed to monitor gradient responses in complex living tissues. Here, we describe a laser-wound assay to generate focal injury in zebrafish larvae and monitor changes in behaviour and cytoskeletal dynamics. The first step is to cross adult fish and collect and rear embryos expressing a relevant fluorescent reporter (for example, Lifeact-mRuby, which labels dynamic actin) to an early larval stage. Subsequently, larvae are mounted and prepared for live imaging and wounding under a two-photon microscope. Finally, the resulting data are processed and used for cell segmentation and quantification of actin dynamics. Altogether, this assay allows the visualisation of cellular dynamics in response to acute injury at high resolution and can be combined with other manipulations, such as genetic or chemical perturbations. Key features • This protocol is designed to trigger laser wound in zebrafish larvae using two-photon intravital microscopy. • The ability to wound while imaging makes it possible to monitor the behaviour and actin changes of the cells immediately after gradient exposure. • The protocol requires a two-photon microscope for best results. Compared with one-photon laser wounding, the injury is more precise and has better tissue penetration. • The focal nature of the wounds is suitable for studies of neutrophil swarming/aggregation and can be further adapted to infectious settings.