Microfluidics and fluorescence microscopy protocol to study the response of C. elegans to chemosensory stimuli.

Here, we present a protocol to use microfluidics in combination with fluorescence microscopy to expose the C. elegans tail to chemosensory stimuli. We describe steps for the preparation of microfluidic chips and sample preparation through the sedation of C. elegans. We detail flow calibration and imaging of C. elegans through fluorescence microscopy to determine their molecular and/or cellular response to chemosensory stimuli. This protocol can also be applied to amphid neurons by inserting the worm in the chip head-first. For complete details on the use and execution of this protocol, please refer to Bruggeman et al. (2022).1.

Differentiated dynamic response in C. elegans chemosensory cilia.

Cilia are membrane-enveloped organelles that protrude from the surface of most eurokaryotic cells and play crucial roles in sensing the external environment. For maintenance and function, cilia are dependent on intraflagellar transport (IFT). Here, we use a combination of microfluidics and fluorescence microscopy to study the response of phasmid chemosensory neurons, in live Caenorhabditis elegans, to chemical stimuli. We find that chemical stimulation results in unexpected changes in IFT and ciliary structure. Notably, stimulation with hyperosmotic solutions or chemical repellents results in different responses, not only in IFT, ciliary structure, and cargo distribution, but also in neuronal activity. The response to chemical repellents results in habituation of the neuronal activity, suggesting that IFT plays a role in regulating the chemosensory response. Our findings show that cilia are able to sense and respond to different external cues in distinct ways, highlighting the flexible nature of cilia as sensing hubs.

Nonlinear mechanics of human mitotic chromosomes.

In preparation for mitotic cell division, the nuclear DNA of human cells is compacted into individualized, X-shaped chromosomes1. This metamorphosis is driven mainly by the combined action of condensins and topoisomerase IIα (TOP2A)2,3, and has been observed using microscopy for over a century. Nevertheless, very little is known about the structural organization of a mitotic chromosome. Here we introduce a workflow to interrogate the organization of human chromosomes based on optical trapping and manipulation. This allows high-resolution force measurements and fluorescence visualization of native metaphase chromosomes to be conducted under tightly controlled experimental conditions. We have used this method to extensively characterize chromosome mechanics and structure. Notably, we find that under increasing mechanical load, chromosomes exhibit nonlinear stiffening behaviour, distinct from that predicted by classical polymer models4. To explain this anomalous stiffening, we introduce a hierarchical worm-like chain model that describes the chromosome as a heterogeneous assembly of nonlinear worm-like chains. Moreover, through inducible degradation of TOP2A5 specifically in mitosis, we provide evidence that TOP2A has a role in the preservation of chromosome compaction. The methods described here open the door to a wide array of investigations into the structure and dynamics of both normal and disease-associated chromosomes.