Rahman MM et al. (2021) Bio Protoc "A Workflow for High-pressure Freezing and Freeze Substitution of the Caenorhabditis elegans Embryo for Ultrastructural Analysis by Conventional and Volume Electron Microscopy."

Chang IY, Narayan K, Rahman MM, Cohen-Fix O

[

Bio Protoc,

2021]

The free-living nematode Caenorhabditis elegans is a popular model system for studying developmental biology. Here we describe a detailed protocol to high-pressure freeze the C. elegans embryo (either ex vivo after dissection, or within the intact worm) followed by quick freeze substitution. Processed samples are suitable for ultrastructural analysis by conventional electron microscopy (EM) or newer volume EM (vEM) approaches such as Focused Ion Beam Scanning Electron Microscopy (FIB-SEM). The ultrastructure of cellular features such as the nuclear envelope, chromosomes, endoplasmic reticulum and mitochondria are well preserved after these experimental procedures and yield accurate 3D models for visualization and analysis ( Chang et al., 2020 ). This protocol was used in the 3D reconstruction of membranes and chromosomes after pronuclear meeting in the C. elegans zygote ( Rahman et al., 2020 ).

Sood P et al. (2017) Traffic "Cargo crowding at actin-rich regions along axons causes local traffic jams."

Kumar TV, Koushika SP, Nonet ML, Murthy K, Menon GI, Sood P

[

Traffic,

2017]

Steady axonal cargo flow is central to the functioning of healthy neurons. However, a substantial fraction of cargo in axons remains stationary up to several minutes (Kang, Tian et al. 2008, Tang, Scott et al. 2012, Tang, Scott et al. 2013, Iacobucci, Rahman et al. 2014). We examine the transport of precursors of synaptic vesicles (pre-SVs), endosomes and mitochondria in C. elegans touch receptor neurons (TRNs), showing that stationary cargo are predominantly present at actin-rich regions along the neuronal process. Stationary vesicles at actin-rich regions increase the propensity of moving vesicles to stall at the same location, resulting in traffic jams arising from physical crowding. Such local traffic jams at actin-rich regions are likely to be a general feature of axonal transport since they also occur in Drosophila neurons. Repeated touch stimulation of C. elegans reduces the density of stationary pre-SVs, indicating that these traffic jams can act as both sources and sinks of vesicles. This suggests that vesicles trapped in actin-rich regions are functional reservoirs that may contribute to maintaining robust cargo flow in the neuron.