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Freshwater planarians normally glide smoothly through ciliary propulsion on their ventral side. Certain environmental conditions, however, can induce musculature-driven forms of locomotion: peristalsis or scrunching. While peristalsis results from a ciliary defect, scrunching is independent of cilia function and is a specific response to certain stimuli, including amputation, noxious temperature, extreme pH, and ethanol. Thus, these two musculature-driven gaits are mechanistically distinct. However, they can be difficult to distinguish qualitatively. Here, we provide a protocol for inducing scrunching using various physical and chemical stimuli. We detail the quantitative characterization of scrunching, which can be used to distinguish it from peristalsis and gliding, using freely available software. Since scrunching is a universal planarian gait, albeit with characteristic species-specific differences, this protocol can be broadly applied to all species of planarians, when using appropriate considerations. To demonstrate this, we compare the response of the two most popular planarian species used in behavioral research, Dugesia japonica and Schmidtea mediterranea, to the same set of physical and chemical stimuli. Furthermore, the specificity of scrunching allows this protocol to be used in conjunction with RNA interference and/or pharmacological exposure to dissect the molecular targets and neuronal circuits involved, potentially providing mechanistic insight into important aspects of nociception and neuromuscular communication.

Within the pericentric regions of human chromosomes reside large arrays of tandemly repeated satellite sequences. Expression of the human pericentric satellite HSATII is prevented by extensive heterochromatin silencing in normal cells, yet in many cancer cells, HSATII RNA is aberrantly expressed and accumulates in large nuclear foci in cis. Expression and aggregation of HSATII RNA in cancer cells is concomitant with recruitment of key chromatin regulatory proteins including methyl-CpG binding protein 2 (MeCP2). While HSATII expression has been observed in a wide variety of cancer cell lines and tissues, the effect of its expression is unknown. We tested the effect of stable expression of HSATII RNA within cells that do not normally express HSATII. Ectopic HSATII expression in HeLa and primary fibroblast cells leads to focal accumulation of HSATII RNA in cis and triggers the accumulation of MeCP2 onto nuclear HSATII RNA bodies. Further, long-term expression of HSATII RNA leads to cell division defects including lagging chromosomes, chromatin bridges, and other chromatin defects. Thus, expression of HSATII RNA in normal cells phenocopies its nuclear accumulation in cancer cells and allows for the characterization of the cellular events triggered by aberrant expression of pericentric satellite RNA. Supplementary Information The online version contains supplementary material available at 10.1007/s00412-021-00753-0.

Protein folding and degradation are both required for protein quality control, an essential cellular activity that underlies normal growth and development. We investigated how BOB1, an Arabidopsis thaliana small heat shock protein, maintains normal plant development. bob1 mutants exhibit organ polarity defects and have expanded domains of KNOX gene expression. Some of these phenotypes are ecotype specific suggesting that other genes function to modify them. Using a genetic approach we identified an interaction between BOB1 and FIL, a gene required for abaxial organ identity. We also performed an EMS enhancer screen using the bob1-3 allele to identify pathways that are sensitized by a loss of BOB1 function. This screen identified genetic, but not physical, interactions between BOB1 and the proteasome subunit RPT2a. Two other proteasome subunits, RPN1a and RPN8a, also interact genetically with BOB1. Both BOB1 and the BOB1-interacting proteasome subunits had previously been shown to interact genetically with the transcriptional enhancers AS1 and AS2, genes known to regulate both organ polarity and KNOX gene expression. Our results suggest a model in which BOB1 mediated protein folding and proteasome mediated protein degradation form a functional proteostasis module required for ensuring normal plant development.

Asexual freshwater planarians reproduce by transverse bisection (binary fission) into two pieces. This process produces a head and a tail, which fully regenerate within 1–2 weeks. How planarians split into two offspring—using only their musculature and substrate traction—is a challenging biomechanics problem. We found that three different species, Dugesia japonica, Girardia tigrina and Schmidtea mediterranea, have evolved three different mechanical solutions to self-bisect. Using time lapse imaging of the fission process, we quantitatively characterize the main steps of division in the three species and extract the distinct and shared key features. Across the three species, planarians actively alter their body shape, regulate substrate traction, and use their muscles to generate tensile stresses large enough to overcome the ultimate tensile strength of the tissue. Moreover, we show that how each planarian species divides dictates how resources are split among its offspring. This ultimately determines offspring survival and reproductive success. Thus, heterospecific differences in the mechanics of self-bisection of individual worms explain the observed differences in the population reproductive strategies of different planarian species.

High-throughput screening (HTS) using new approach methods is revolutionizing toxicology. Asexual freshwater planarians are a promising invertebrate model for neurotoxicity HTS because their diverse behaviors can be used as quantitative readouts of neuronal function. Currently, three planarian species are commonly used in toxicology research: Dugesia japonica, Schmidtea mediterranea, and Girardia tigrina. However, only D. japonica has been demonstrated to be suitable for HTS. Here, we assess the two other species for HTS suitability by direct comparison with D. japonica. Through quantitative assessments of morphology and multiple behaviors, we assayed the effects of 4 common solvents (DMSO, ethanol, methanol, ethyl acetate) and a negative control (sorbitol) on neurodevelopment. Each chemical was screened blind at 5 concentrations at two time points over a twelve-day period. We obtained two main results: First, G. tigrina and S. mediterranea planarians showed significantly reduced movement compared to D. japonica under HTS conditions, due to decreased health over time and lack of movement under red lighting, respectively. This made it difficult to obtain meaningful readouts from these species. Second, we observed species differences in sensitivity to the solvents, suggesting that care must be taken when extrapolating chemical effects across planarian species. Overall, our data show that D. japonica is best suited for behavioral HTS given the limitations of the other species. Standardizing which planarian species is used in neurotoxicity screening will facilitate data comparisons across research groups and accelerate the application of this promising invertebrate system for first-tier chemical HTS, helping streamline toxicology testing.

In response to noxious stimuli, planarians cease their typical ciliary gliding and exhibit an oscillatory type of locomotion called scrunching. We have previously characterized the biomechanics of scrunching and shown that it is induced by specific stimuli, such as amputation, noxious heat, and extreme pH. Because these specific inducers are known to activate Transient Receptor Potential (TRP) channels in other systems, we hypothesized that TRP channels control scrunching. We found that chemicals known to activate TRPA1 (allyl isothiocyanate (AITC) and hydrogen peroxide) and TRPV (capsaicin and anandamide) in other systems induce scrunching in the planarian species Dugesia japonica and, except for anandamide, in Schmidtea mediterranea. To confirm that these responses were specific to either TRPA1 or TRPV, respectively, we tried to block scrunching using selective TRPA1 or TRPV antagonists and RNA interference (RNAi) mediated knockdown. Unexpectedly, co-treatment with a mammalian TRPA1 antagonist, HC-030031, enhanced AITC-induced scrunching by decreasing the latency time, suggesting an agonistic relationship in planarians. We further confirmed that TRPA1 in both species is necessary for AITC-induced scrunching using RNAi. Conversely, while co-treatment of a mammalian TRPV antagonist, SB-366791, also enhanced capsaicin-induced reactions in D. japonica, combined knockdown of two previously identified D. japonica TRPV genes (DjTRPVa and DjTRPVb) did not inhibit capsaicin-induced scrunching. Surprisingly, RNAi of either DjTRPAa or DjTRPVa/DjTRPVb disrupted scrunching induced by the endocannabinoid and TRPV agonist, anandamide. Overall, our results show that although scrunching induction can involve different initial pathways for sensing stimuli, this behavior’s signature dynamical features are independent of the inducer, implying that scrunching is a stereotypical planarian escape behavior in response to various noxious stimuli that converge on a single downstream pathway. Understanding which aspects of nociception are conserved or not across different organisms can provide insight into the underlying regulatory mechanisms to better understand pain sensation.

Background High temperature stress responses are vital for plant survival. The mechanisms that plants use to sense high temperatures are only partially understood and involve multiple sensing and signaling pathways. Here we describe the development of the RootScope, an automated microscopy system for quantitating heat shock responses in plant roots. Results The promoter of Hsp17.6 was used to build a Hsp17.6 p:GFP transcriptional reporter that is induced by heat shock in Arabidopsis. An automated fluorescence microscopy system which enables multiple roots to be imaged in rapid succession was used to quantitate Hsp17.6 p:GFP response dynamics. Hsp17.6 p:GFP signal increased with temperature increases from 28°C to 37°C. At 40°C the kinetics and localization of the response are markedly different from those at 37°C. This suggests that different mechanisms mediate heat shock responses above and below 37°C. Finally, we demonstrate that Hsp17.6 p:GFP expression exhibits wave like dynamics in growing roots. Conclusions The RootScope system is a simple and powerful platform for investigating the heat shock response in plants.

Genome-wide studies have identified abundant small, noncoding RNAs, including small nuclear RNAs, small nucleolar RNAs (snoRNAs), cryptic unstable transcripts (CUTs), and upstream regulatory RNAs (uRNAs), that are transcribed by RNA polymerase II (pol II) and terminated by an Nrd1-dependent pathway. Here, we show that the prolyl isomerase Ess1 is required for Nrd1-dependent termination of noncoding RNAs. Ess1 binds the carboxy-terminal domain (CTD) of pol II and is thought to regulate transcription by conformational isomerization of Ser-Pro bonds within the CTD. In ess1 mutants, expression of approximately 10% of the genome was altered, due primarily to defects in termination of snoRNAs, CUTs, stable unannotated transcripts, and uRNAs. Ess1 promoted dephosphorylation of Ser5 (but not Ser2) within the CTD, most likely by the Ssu72 phosphatase. We also provide evidence for a competition between Nrd1 and Pcf11 for CTD binding that is regulated by Ess1. These data indicate that a prolyl isomerase is required for specifying the "CTD code."