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).

Replication Data for:A small excitation window allows long-duration single-molecule imaging, with reduced background autofluorescence, in C. elegans neurons

The dataset contains the data and the MATLAB scripts associated with publication titled A small excitation window allows long-duration single-molecule imaging, with reduced background autofluorescence, in C. elegans neurons. Here we provide scripts and data corresponding to three different aspects: (1) the characterization of the technique, small-window illumination microscopy (SWIM), that was developed in this work (2) the numerical simulations to explain how SWIM reduces the amount of out-of-focus autofluoresence background during imaging. (3) the statistical analysis of the motion of vesicles moving back-and-forth in a C. elegans dendrite, imaged using SWIM. The data and codes are orgainized in separate folders, with the titles indicating the figure the data belongs to. In addition, there are a couple of folders containing essential scripts used for analysis of single-molecule tracks. Each folder contains a ReadMe.txt file which details the data (and structure) and the accompanying script(s). Data is stored as .csv (or xlsx) files and scripts are written in MATLAB (The Math Works, Inc., R2021a)

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

Publisher Correction: Nonlinear mechanics of human mitotic chromosomes

In the version of this article initially published, Extended Data Fig. 5 was a duplicate of Extended Data Fig. 6. The correct image is now in place in the HTML and PDF versions of the article

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