Analysis of nematode mechanics by piezoresistive displacement clamp

Abstract

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 m 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.biomechanics ͉ Caenorhabditis elegans ͉ microelectromechanical systems A nalyses of the nematode Caenorhabditis elegans and its mutants enable systematic study of the interplay between genes and behaviors that range from simple movement to successful mating. C. elegans and other nematodes move in a sinusoidal fashion by generating waves of alternating dorsal and ventral muscle contraction. These waves of muscle contraction produce local bending in the cuticle, which is opposed by a high hydrostatic pressure (1, 2). In the laboratory and in the natural soil environment, C. elegans crawls across surfaces in a thin layer of moisture. The transformation of signals in the neuromuscular plant into behavior is constrained by body mechanics. Similarly, body mechanics determines how loads applied to the outer body surface are conveyed to mechanosensory neurons. To learn more, we developed a piezoresistive (PR)-based system with force and displacement ranges that are unavailable with existing methods.The nematode body plan consists of an outer tube separated from an inner tube by a fluid-filled pseudocoelom (Fig. 1). The cuticle, hypodermis, excretory system, neurons, and longitudinal muscles comprise the outer tube or shell, and the pharynx, intestine, and gonad form the inner tube (3). Internal tissues are under pressures on the order of 2-30 kPa (1), suggesting that nematodes have a shell-type hydrostatic skeleton. Very little is known about the relative importance of hydrostatic pressure and cuticle structure and elasticity to overall b...

Worms under Pressure: Bulk Mechanical Properties of C. elegans Are Independent of the Cuticle

“…the local material properties of the worm have confirmed that Dpy worms exhibit an~25% softer cuticle (19), consistent with a significant disruption of cuticle structure. However, we found that both dpy-5 and dpy-10 worms exhibited no significant difference in their bulk modulus compared with wild-type worms (Fig.…”

“…Because the worm is approximately radially symmetric, we treat the worm as a cylinder (14,19,37) and estimate changes in the body volume from its area and the length, using VðpÞ ¼ ðp=4ÞA 2 ðpÞ=LðpÞ:…”

“…The modulus is determined by internal structures and not the cuticle Previous studies have concluded that the collagen-rich cuticle plays an important role in determining the local stiffness (19,20) and bending rigidity (26) of worms. To test the role of the cuticle, worms were exposed to proteinase K, an enzyme that degrades cuticle proteins (22,42).…”

“…Importantly, this does not imply that the cuticle is a mechanically soft material. The cuticle is known to be comprised of a dense collagenous A B shell, and previous work using local mechanical deformation experiments has suggested that it has a very high stiffness on the order of 257-457 MPa (19,20). However, it is known to be very thin (0.5 mm, roughly 2% of the diameter of the worm), and thus its contribution to the overall stiffness of the worm need not be significant (42).…”

“…Local stiffness measurements of the C. elegans cuticle have been performed using a piezoelectric transducer, which indicate that the cuticle exhibits a high elastic modulus in the range of 380 MPa (19). A similar local stiffness study using the buckling of nanowires attached to an atomic force microscope reported an elastic modulus of 457 MPa on the lateral alae, and 257 MPa in the region immediately around the lateral alae (20).…”