Park SJ et al. (2011) Rev Sci Instrum "Piezoresistive cantilever force-clamp system."

Petzold BC, Pruitt BL, Goodman MB, Park SJ

[Rev Sci Instrum, 2011]

We present a microelectromechanical device-based tool, namely, a force-clamp system that sets or "clamps" the scaled force and can apply designed loading profiles (e.g., constant, sinusoidal) of a desired magnitude. The system implements a piezoresistive cantilever as a force sensor and the built-in capacitive sensor of a piezoelectric actuator as a displacement sensor, such that sample indentation depth can be directly calculated from the force and displacement signals. A programmable real-time controller operating at 100 kHz feedback calculates the driving voltage of the actuator. The system has two distinct modes: a force-clamp mode that controls the force applied to a sample and a displacement-clamp mode that controls the moving distance of the actuator. We demonstrate that the system has a large dynamic range (sub-nN up to tens of N force and nm up to tens of m displacement) in both air and water, and excellent dynamic response (fast response time, <2 ms and large bandwidth, 1 Hz up to 1 kHz). In addition, the system has been specifically designed to be integrated with other instruments such as a microscope with patch-clamp electronics. We demonstrate the capabilities of the system by using it to calibrate the stiffness and sensitivity of an electrostatic actuator and to measure the mechanics of a living, freely moving Caenorhabditis elegans nematode.

Park SJ et al. (2007) Proc Natl Acad Sci U S A "Analysis of nematode mechanics by piezoresistive displacement clamp."

Pruitt BL, Goodman MB, Park SJ

[Proc Natl Acad Sci U S A, 2007]

Studying animal mechanics is critical for understanding how signals in the neuromuscular system give rise to behavior and how force-sensing organs and sensory neurons work. Few techniques exist to provide forces and displacements appropriate for such studies. To address this technological gap, we developed a metrology using piezoresistive cantilevers as force-displacement sensors coupled to a feedback system to apply and maintain defined load profiles to micrometer-scale animals. We show that this system can deliver forces between 10(-8) and 10(-3) N across distances of up to 100 mum with a resolution of 12 nN between 0.1 Hz and 100 kHz. We use this new metrology to show that force-displacement curves of wild-type nematodes (Caenorhabditis elegans) are linear. Because nematodes have approximately cylindrical bodies, this finding demonstrates that nematode body mechanics can be modeled as a cylindrical shell under pressure. Little is known about the relative importance of hydrostatic pressure and shell mechanics, however. We show that dissipating pressure by cuticle puncture or decreasing it by hyperosmotic shock has only a modest effect on stiffness, whereas defects in the dpy-5 and lon-2 genes, which alter body shape and cuticle proteins, decrease and increase stiffness by 25% and 50%, respectively. This initial analysis of C. elegans body mechanics suggests that shell mechanics dominates stiffness and is a first step in understanding how body mechanics affect locomotion and force sensing.

Jeong A et al. (2023) J Hazard Mater "Nanoplastics exacerbate Parkinson's disease symptoms in C. elegans and human cells."

Jeong A, Kim KW, Lee EJ, Park SJ

[J Hazard Mater, 2023]

The increasing prevalence of nanoplastics in our environment due to the widespread use of plastics poses potential health risks that are not yet fully understood. This study examines the physiological and neurotoxic effects of these minuscule nanoplastic particles on the nematode Caenorhabditis elegans as well as on human cells. Here, we find that 25 nm polystyrene nanoplastic particles can inhibit animal growth and movement at very low concentrations, with varying effects on their surface groups. Furthermore, these nanoplastic particles not only accumulate in the digestive tract but also penetrate further into extraintestinal tissues. Such nanoplastics significantly compromise the integrity of the intestinal barrier, leading to "leaky gut" conditions and cause mitochondrial fragmentation in muscles, which possibly explains the observed movement impairments. A striking discovery was that these nanoplastics exacerbate symptoms similar to those of Parkinson's disease (PD), including dopaminergic neuronal degeneration, locomotor dysfunction, and accumulation of α-Synuclein aggregates. Importantly, our study demonstrates that the detrimental effects of nanoplastics on the aggregation of α-Synuclein extend to both C. elegans and human cell models of PD. In conclusion, our research highlights the potential health hazards linked to the physicochemical properties of nanoplastics, underlining the urgency of understanding their interactions with biological systems. ENVIRONMENTAL IMPLICATION: The escalating prevalence of nanoplastics in the environment due to widespread plastic usage raises potential health risks. Studies conducted on C. elegans indicate that even low concentrations of 25 nm polystyrene nanoplastics can impair growth and movement. These particles accumulate in the digestive system, compromising the intestinal barrier, causing "leaky gut", as well as inducing Parkinson's-like symptoms. Importantly, in both C. elegans and human cell models of Parkinson's disease, such nanoplastics penetrate tissues or cells and increase α-Synuclein aggregates. This underscores the urgent need to understand the interactions of nanoplastics with biological systems and highlights potential environmental and health consequences.

Petzold BC et al. (2013) Integr Biol (Camb) "MEMS-based force-clamp analysis of the role of body stiffness in C. elegans touch sensation."

Mazzochette EA, Pruitt BL, Park SJ, Petzold BC, Goodman MB

[Integr Biol (Camb), 2013]

Touch is enabled by mechanoreceptor neurons in the skin and plays an essential role in our everyday lives, but is among the least understood of our five basic senses. Force applied to the skin deforms these neurons and activates ion channels within them. Despite the importance of the mechanics of the skin in determining mechanoreceptor neuron deformation and ultimately touch sensation, the role of mechanics in touch sensitivity is poorly understood. Here, we use the model organism Caenorhabditis elegans to directly test the hypothesis that body mechanics modulate touch sensitivity. We demonstrate a microelectromechanical system (MEMS)-based force clamp that can apply calibrated forces to freely crawling C. elegans worms and measure touch-evoked avoidance responses. This approach reveals that wild-type animals sense forces <1 N and indentation depths <1 m. We use both genetic manipulation of the skin and optogenetic modulation of body wall muscles to alter body mechanics. We find that small changes in body stiffness dramatically affect force sensitivity, while having only modest effects on indentation sensitivity. We investigate the theoretical body deformation predicted under applied force and conclude that local mechanical loads induce inward bending deformation of the skin to drive touch sensation in C. elegans.

Kim SK et al. (2018) Environ Microbiol "Bacterial ornithine lipid, a surrogate membrane lipid under phosphate-limiting conditions, plays important roles in bacterial persistence and interaction with host."

Kim SK, Choi YS, Im DS, Li XH, Lee JH, Park SJ

[Environ Microbiol, 2018]

Ornithine lipids (OLs) are bacteria-specific lipids that are found in the outer membrane of Gram (-) bacteria and increase as surrogates of phospholipids under phosphate-limited environmental conditions. We investigated the effects of OL increase in bacterial membranes on pathogen virulence and the host immune response. In Pseudomonas aeruginosa, we increased OL levels in membranes by overexpressing the OL-synthesizing operon (olsBA). These increases changed the bacterial surface charge and hydrophobicity, which reduced bacterial susceptibility to antibiotics and antimicrobial peptides (AMPs), interfered with the binding of macrophages to bacterial cells and enhanced bacterial biofilm formation. When grown under low phosphate conditions, P. aeruginosa became more persistent in the treatment of antibiotics and AMPs in an olsBA-dependent manner. While OLs increased persistence, they attenuated P. aeruginosa virulence; in host cells, they reduced the production of inflammatory factors (iNOS, COX-2, PGE₂ and nitric oxide) and increased intracellular Ca²⁺ release. Exogenously added OL had similar effects on P. aeruginosa and host cells. Our results suggest that bacterial OL plays important roles in bacteria-host interaction in a way that enhances bacterial persistence and develops chronic adaptation to infection.

Eastwood AL et al. (2015) Proc Natl Acad Sci U S A "Tissue mechanics govern the rapidly adapting and symmetrical response to touch."

Vergassola M, Sanzeni A, Eastwood AL, Petzold BC, Goodman MB, Pruitt BL, Park SJ

[Proc Natl Acad Sci U S A, 2015]

Interactions with the physical world are deeply rooted in our sense of touch and depend on ensembles of somatosensory neurons that invade and innervate the skin. Somatosensory neurons convert the mechanical energy delivered in each touch into excitatory membrane currents carried by mechanoelectrical transduction (MeT) channels. Pacinian corpuscles in mammals and touch receptor neurons (TRNs) in Caenorhabditis elegans nematodes are embedded in distinctive specialized accessory structures, have low thresholds for activation, and adapt rapidly to the application and removal of mechanical loads. Recently, many of the protein partners that form native MeT channels in these and other somatosensory neurons have been identified. However, the biophysical mechanism of symmetric responses to the onset and offset of mechanical stimulation has eluded understanding for decades. Moreover, it is not known whether applied force or the resulting indentation activate MeT channels. Here, we introduce a system for simultaneously recording membrane current, applied force, and the resulting indentation in living C. elegans (Feedback-controlled Application of mechanical Loads Combined with in vivo Neurophysiology, FALCON) and use it, together with modeling, to study these questions. We show that current amplitude increases with indentation, not force, and that fast stimuli evoke larger currents than slower stimuli producing the same or smaller indentation. A model linking body indentation to MeT channel activation through an embedded viscoelastic element reproduces the experimental findings, predicts that the TRNs function as a band-pass mechanical filter, and provides a general mechanism for symmetrical and rapidly adapting MeT channel activation relevant to somatosensory neurons across phyla and submodalities.

Petzold BC et al. (2011) Biophys J "Caenorhabditis elegans body mechanics are regulated by body wall muscle tone."

Ponce P, Powell C, Roozeboom C, Goodman MB, Park SJ, Petzold BC, Pruitt BL

[Biophys J, 2011]

Body mechanics in the nematode Caenorhabditis elegans are central to both mechanosensation and locomotion. Previous work revealed that the mechanics of the outer shell, rather than internal hydrostatic pressure, dominates stiffness. This shell is comprised of the cuticle and the body wall muscles, either of which could contribute to the body mechanics. Here, we tested the hypothesis that the muscles are an important contributor by modulating muscle tone using optogenetic and pharmacological tools, and measuring animal stiffness using piezoresistive microcantilevers. As a proxy for muscle tone, we measured changes in animal length under the same treatments. We found that treatments that induce muscle contraction generally resulted in body shortening and stiffening. Conversely, methods to relax the muscles more modestly increased length and decreased stiffness. The results support the idea that body wall muscle activation contributes significantly to and can modulate C.elegans body mechanics. Modulation of body stiffness would enable nematodes to tune locomotion or swimming gaits and may have implications in touch sensation.

Kim J et al. (2015) PLoS Genet "The Evolutionarily Conserved LIM Homeodomain Protein LIM-4/LHX6 Specifies the Terminal Identity of a Cholinergic and Peptidergic C. elegans Sensory/Inter/Motor Neuron-Type."

Park SJ, Huh YH, Kim K, Kweon HS, Kim MO, Yeon J, Li C, Kim J, Fang Z, Kang K, Choi SK, Ryoo ZY, Jeon WB

[PLoS Genet, 2015]

The expression of specific transcription factors determines the differentiated features of postmitotic neurons. However, the mechanism by which specific molecules determine neuronal cell fate and the extent to which the functions of transcription factors are conserved in evolution are not fully understood. In C. elegans, the cholinergic and peptidergic SMB sensory/inter/motor neurons innervate muscle quadrants in the head and control the amplitude of sinusoidal movement. Here we show that the LIM homeobox protein LIM-4 determines neuronal characteristics of the SMB neurons. In lim-4 mutant animals, expression of terminal differentiation genes, such as the cholinergic gene battery and the flp-12 neuropeptide gene, is completely abolished and thus the function of the SMB neurons is compromised. LIM-4 activity promotes SMB identity by directly regulating the expression of the SMB marker genes via a distinct cis-regulatory motif. Two human LIM-4 orthologs, LHX6 and LHX8, functionally substitute for LIM-4 in C. elegans. Furthermore, C. elegans LIM-4 or human LHX6 can induce cholinergic and peptidergic characteristics in the human neuronal cell lines. Our results indicate that the evolutionarily conserved LIM-4/LHX6 homeodomain proteins function in generation of precise neuronal subtypes.

Rudel D et al. (2005) Dev Biol "Gonadogenesis in Pristionchus pacificus and organ evolution: development, adult morphology and cell-cell interactions in the hermaphrodite gonad."

Rudel D, Riebesell M, Sommer RJ

[Dev Biol, 2005]

The nematode gonad is an exemplary system for the study of organogenesis and fundamental problems in developmental and cellular biology. Nematode gonads vary dramatically across species (Chitwood, B.G., Chitwood, M.B., 1950. Introduction to Nematology." University Park Press, Baltimore; Felix, M.A., Sternberg, P.W., 1996. Symmetry breakage in the development of one-armed gonads in nematodes. Development 122, 2129-2142). As such, comparative developmental biology of gonadogenesis offers the potential to investigate changes in developmental and cellular processes that result in novel organ morphologies and thus may give insights into how these changes can affect animal bauplane. Pristionchus pacificus is a free-living nematode that diverged from the model nematode Caenorhabditis elegans around 200-300 million years ago. The morphology and development of P. pacificus is highly homologous to that of C. elegans. However, many differences in morphology and the underlying molecular signaling networks are easy to identify, making P. pacificus ideal for a comparative approach. Here, we report a detailed description of the P. pacificus hermaphrodite gonad using electron and fluorescent microscopy that will provide a basis for both phenotypic studies of genetic mutations and in vivo molecular studies of cloned genes involved in P. pacificus gonad development. We report that the morphology of the P. pacificus gonad is distinct from that of C. elegans. Among these differences are germ line patterning differences, heterochronic differences, novel gonadal arm-migrations, novel cellular composition of some somatic tissues (e.g., the number of cells that comprise the sheath and different spermathecal regions are different), the absence of a somatic tissue (e.g., the spermathecal valve cells), a novel architecture for the sheath, and changes in the cellular and sub-cellular morphology of the individual sheath cells. Additionally, we report a set of cell ablations in P. pacificus that indicate extensive cell communication between the somatic gonadal tissues and the germ line. Individual ablation experiments in P. pacificus show significant differences in the effects of individual somatic tissues on germ line patterning in comparison to C. elegans.

Glock C et al. (2015) Methods Mol Biol "Microbial Rhodopsin Optogenetic Tools: Application for Analyses of Synaptic Transmission and of Neuronal Network Activity in Behavior."

Glock C, Gottschalk A, Nagpal J

[Methods Mol Biol, 2015]

Optogenetics was introduced as a new technology in the neurosciences about a decade ago (Zemelman et al., Neuron 33:15-22, 2002; Boyden et al., Nat Neurosci 8:1263-1268, 2005; Nagel et al., Curr Biol 15:2279-2284, 2005; Zemelman et al., Proc Natl Acad Sci USA 100:1352-1357, 2003). It combines optics, genetics, and bioengineering to render neurons sensitive to light, in order to achieve a precise, exogenous, and noninvasive control of membrane potential, intracellular signaling, network activity, or behavior (Rein and Deussing, Mol Genet Genomics 287:95-109, 2012; Yizhar et al., Neuron 71:9-34, 2011). As C. elegans is transparent, genetically amenable, has a small nervous system mapped with synapse resolution, and exhibits a rich behavioral repertoire, it is especially open to optogenetic methods (White et al., Philos Trans R Soc Lond B Biol Sci 314:1-340, 1986; De Bono et al., Optogenetic actuation, inhibition, modulation and readout for neuronal networks generating behavior in the nematode Caenorhabditis elegans, In: Hegemann P, Sigrist SJ (eds) Optogenetics, De Gruyter, Berlin, 2013; Husson et al., Biol Cell 105:235-250, 2013; Xu and Kim, Nat Rev Genet 12:793-801, 2011). Optogenetics, by now an "exploding" field, comprises a repertoire of different tools ranging from transgenically expressed photo-sensor proteins (Boyden et al., Nat Neurosci 8:1263-1268, 2005; Nagel et al., Curr Biol 15:2279-2284, 2005) or cascades (Zemelman et al., Neuron 33:15-22, 2002) to chemical biology approaches, using photochromic ligands of endogenous channels (Szobota et al., Neuron 54:535-545, 2007). Here, we will focus only on optogenetics utilizing microbial rhodopsins, as these are most easily and most widely applied in C. elegans. For other optogenetic tools, for example the photoactivated adenylyl cyclases (PACs, that drive neuronal activity by increasing synaptic vesicle priming, thus exaggerating rather than overriding the intrinsic activity of a neuron, as occurs with rhodopsins), we refer to other literature (Weissenberger et al., J Neurochem 116:616-625, 2011; Steuer Costa et al., Photoactivated adenylyl cyclases as optogenetic modulators of neuronal activity, In: Cambridge S (ed) Photswitching proteins, Springer, New York, 2014). In this chapter, we will give an overview of rhodopsin-based optogenetic tools, their properties and function, as well as their combination with genetically encoded indicators of neuronal activity. As there is not "the" single optogenetic experiment we could describe here, we will focus more on general concepts and "dos and don'ts" when designing an optogenetic experiment. We will also give some guidelines on which hardware to use, and then describe a typical example of an optogenetic experiment to analyze the function of the neuromuscular junction, and another application, which is Ca(2+) imaging in body wall muscle, with upstream neuronal excitation using optogenetic stimulation. To obtain a more general overview of optogenetics and optogenetic tools, we refer the reader to an extensive collection of review articles, and in particular to volume 1148 of this book series, "Photoswitching Proteins."