Biology subjects

Lesanpezeshki, L.

Publications and source records attributed to Lesanpezeshki, L..

5 recordsLinked to original sources

bioRxiv2024

FHOD-1/profilin-mediated actin assembly protect sarcomeres against contraction-induced deformation in C. elegans

Formin HOmology Domain 2-containing (FHOD) proteins are a subfamily of actin-organizing formins important for striated muscle development in many animals. We showed previously that absence of the sole FHOD protein, FHOD-1, from C. elegans results in thin body-wall muscles with misshapen dense bodies that serve as sarcomere Z-lines. We demonstrate here that actin polymerization by FHOD-1 is required for its function in muscle development, and that FHOD-1 cooperates with profilin PFN-3 for dense body morphogenesis, and profilins PFN-2 and PFN-3 to promote body-wall muscle growth. We further demonstrate dense bodies in fhod-1 and pfn-3 mutants are less stable than in wild type animals, having a higher proportion of dynamic protein, and becoming distorted by prolonged muscle contraction. We also observe accumulation of actin depolymerization factor/cofilin homolog UNC-60B in body-wall muscle of these mutants. Such accumulations may indicate targeted disassembly of thin filaments dislodged from unstable dense bodies, and may account for the abnormally slow growth and reduced strength of body-wall muscle in fhod-1 mutants. Overall, these results show the importance of FHOD protein-mediated actin assembly to forming stable sarcomere Z-lines, and identify profilin as a new contributor to FHOD activity in striated muscle development.

cell biology↗

bioRxiv2022

Functional imaging and quantification of multi-neuronal olfactory responses in C. elegans

Many animals perceive odorant molecules by collecting information from ensembles of olfactory neurons. Each neuron employs receptors that are tuned to recognize certain odorant molecules by chemical binding affinity. Olfactory systems are able, in principle, to detect and discriminate diverse odorants by using combinatorial coding strategies. Multineuronal imaging with high-throughput stimulus delivery allows comprehensive measurement of ensemble-level sensory representations. We have used microfluidics and multineuronal imaging to study ensemble-level olfactory representations at the sensory periphery of the nematode C. elegans. The collective activity of nematode chemosensory neurons reveals high-dimensional representations of olfactory information across a broad space of odorant molecules. We reveal diverse tuning properties and dose-response curves across chemosensory neurons and across odorants. We describe the unique contribution of each sensory neuron to an ensemble-level code for volatile odorants. We also show how natural stimuli, a set of nematode pheromones, are encoded by the sensory periphery. The integrated activity of the C. elegans chemosensory neurons contains sufficient information to robustly encode the intensity and identity of diverse chemical stimuli.

neuroscience↗

bioRxiv2021

Investigating the correlation of muscle function tests and sarcomere organization in C. elegans

BackgroundCaenorhabditis elegans has been widely used as a model to study muscle structure and function due to many genes having human homologs. Its body wall muscle is functionally and structurally similar to vertebrate skeletal muscle with conserved molecular pathways contributing to sarcomere structure, and muscle function. However, a systematic investigation of the relationship between muscle force and sarcomere organization is lacking. Here, we investigate the contribution of various sarcomere proteins and membrane attachment components to muscle structure and function to introduce C. elegans as a model organism to study the genetic basis of muscle strength. MethodsWe employ two recently developed assays that involve exertion of muscle forces to investigate the correlation of muscle function to sarcomere organization. We utilized a microfluidic pillar-based platform called NemaFlex that quantifies the maximum exertable force and a burrowing assay that challenges the animals to move in three dimensions under a chemical stimulus. We selected 20 mutants with known defects in various substructures of sarcomeres and compared the physiological function of muscle proteins required for force generation and transmission. We also characterized the degree of sarcomere disorganization using immunostaining approaches. ResultsWe find that mutants with genetic defects in thin filaments, thick filaments and M-lines are generally weaker, and our assays are successful in detecting the functional changes in response to each sarcomere location tested. We find that the NemaFlex and burrowing assays are functionally distinct informing on different aspects of muscle physiology. Specifically, the burrowing assay has a larger bandwidth in phenotyping muscle mutants, because it could pick ten additional mutants impaired while exerting normal muscle force in NemaFlex. This enabled us to combine their readouts to develop an integrated muscle function score that was found to correlate with the score for muscle structure disorganization. ConclusionsOur results highlight the suitability of NemaFlex and burrowing assays for evaluating muscle physiology of C. elegans. Using these approaches, we discuss the importance of the studied sarcomere proteins for muscle function and structure. The scoring methodology we have developed lays the foundation for investigating the contribution of conserved sarcomere proteins and membrane attachment components to human muscle function and strength.

physiology↗

bioRxiv2020

A Region of UNC-89 (obscurin) Lying Between Two Protein Kinase Domains is a Highly Elastic Spring Required for Proper Sarcomere Assembly

In C. elegans, unc-89 encodes a set of giant multi-domain proteins (up 8,081 residues) localized to the M-lines of muscle sarcomeres and required for normal sarcomere organization and whole-animal locomotion. Multiple UNC-89 isoforms contain two protein kinase domains. There is conservation in arrangement of domains between UNC-89 and its two mammalian homologs, obscurin and SPEG: kinase, a non-domain region of 647-742 residues, Ig domain, Fn3 domain and a second kinase domain. In all three proteins, this non-domain "interkinase region" has low sequence complexity, high proline content and lacks predicted secondary structure. We report that a major portion of this interkinase (571 residues out of 647 residues) when examined by single molecule force spectroscopy in vitro displays the properties of a random coil and acts as an entropic spring. We used CRISPR/Cas9 to create nematodes carrying an in-frame deletion of the same 571-residue portion of the interkinase. These animals express normal levels of giant internally deleted UNC-89 proteins, and yet show severe disorganization of all portions of the sarcomere in body wall muscle. Super-resolution microscopy reveals extra, short-A-bands lying close to the outer muscle cell membrane and between normally spaced A-bands. Nematodes with this in-frame deletion show defective locomotion and muscle force generation. We designed our CRISPR-generated in-frame deletion to contain an HA tag at the N-terminus of the large UNC-89 isoforms. This HA tag results in normal organization of body wall muscle, but dis-organization of pharyngeal muscle, small body size, and reduced muscle force, likely due to poor nutritional uptake. HighlightsO_LIThe giant muscle proteins UNC-89 and its mammalian homologs have an [~]700 aa non-domain region lying between two protein kinase domains C_LIO_LIBy single molecule force spectroscopy UNC-89 non-domain region is an elastic random coil C_LIO_LINematodes lacking this non-domain region have disorganized sarcomeres and reduced whole animal locomotion C_LIO_LIUNC-89 non-domain region is required for proper assembly of A-bands from thick filaments C_LI

cell biology↗

bioRxiv2019

Pluronic gel-based burrowing assay for rapid assessment of neuromuscular health in C. elegans

Whole-organism phenotypic assays are central to the assessment of neuromuscular function and health in model organisms such as the nematode C. elegans. In this study, we report a new assay format for engaging C. elegans in burrowing that enables rapid assessment of nematode neuromuscular health. In contrast to agar environments that pose specific drawbacks for characterization of C. elegans burrowing ability, here we use the optically transparent and biocompatible Pluronic F-127 gel that transitions from liquid to gel at room temperature, enabling convenient and safe handling of animals. The burrowing assay methodology involves loading animals at the bottom of well plates, casting a liquid-phase of Pluronic on top that solidifies via a modest temperature upshift, enticing animals to reach the surface via chemotaxis to food, and quantifying the relative success animals have in reaching the chemoattractant. We study the influence of Pluronic concentration, gel height and chemoattractant choice to optimize assay performance. To demonstrate the simplicity of the assay workflow and versatility, we show its novel application in multiple areas including (i) evaluating muscle mutants with defects in dense bodies and/or M-lines (pfn-3, atn-1, uig-1, dyc-1, zyx-1, unc-95 and tln-1), (ii) tuning assay conditions to reveal changes in the mutant gei- 8, (iii) sorting of fast burrowers in a genetically-uniform wild-type population for later quantitation of their distinct muscle gene expression, and (iv) testing proteotoxic animal models of Huntington and Parkinsons disease. Results from our studies show that stimulating animals to navigate in a dense environment that offers mechanical resistance to three- dimensional locomotion challenges the neuromuscular system in a manner distinct from standard crawling and thrashing assays. Our simple and high throughput burrowing assay can provide insight into molecular mechanisms for maintenance of neuromuscular health and facilitate screening for therapeutic targets.

neuroscience↗