LM Frisse et al. (1999) International C. elegans Meeting "Origin and evolution of microsatellites in C. elegans"

M Lynch, WK Thomas, L Vassilieva, LM Frisse

[International C. elegans Meeting, 1999]

Microsatellites, small direct repeats, are a ubiquitous feature of eukaryotic genomes. The high mutation rate of microsatellite loci has made them useful in genetic mapping and evolutionary studies. Microsatellites have also been recognized as the causal agents of numerous human genetic diseases and are used as indicators for tumor formation. Despite their widespread use, little is known about the origin of microsatellites, factors that affect microsatellite frequency and distribution, and the rates and patterns of microsatellite mutation. Understanding the rates and patterns of microsatellite evolution is critical for their use in studies of evolution, genetic mapping and mutational mechanisms. The recent completion of the Caenorhabditis elegans genome allows the testing of many of the hypotheses concerning microsatellite evolution. We have identified 953 microsatellites with 2-5 bp repeats, which are at least 10 perfect repeat units in length. These loci have been physically mapped allowing us to investigate the frequency and distribution of microsatellites across an entire metazoan genome. The majority of the loci identified are dimers. There is no clear pattern for microsatellite distribution within the genome. Microsatellite frequencies are not consistent with base composition or dinucleotide sequence composition; however, there is some evidence that microsatellites may be seeded by a telomeric activity. In order to understand the rates and patterns of microsatellite mutations, we assayed 29 microsatellite loci in a set of 80 mutation accumulation lines of the nematode C. elegans propagated for 140 generations. Investigation of mutation rate included dimer loci over a wide range of allele sizes. This study clearly demonstrates that the mutation rate increases with increased repeat number and that the pattern of mutation is biased towards small additions.

Frisse LM et al. (1995) International C. elegans Meeting "EVALUATION OF A MOLECULAR DATABASE FOR NEMATODE TAXONOMY"

Frisse LM, Vida JT, Thomas WK

[International C. elegans Meeting, 1995]

A large number of nematode species remain to be identified. To overcome this problem a common set of taxonomic characters is needed. Nematode species exhibit great diversity at the molecular level. This molecular variation may be useful as a taxonomic tool. Recent technical advances, in particular the advent of the polymerase chain reaction and automated sequencing, have made the generation of sequences for large numbers of individuals a realistic goal. In an attempt to set up a molecular database, we have chosen the D3 expansion segment of the gene for the largest subunit of ribosomal RNA as our molecular species tag. This segment amplifies in a wide range of nematode taxa, can be sequenced from all developmental stages, and provides unambiguous character sets that require no subjective evaluation. When a complete sequence is obtained for each new sample, it is compared to existing sequences for identification. If the sequence represents a new sample it is added to the main database. The existing database contains over 80 sequences and is being evaluated for its usefulness as a taxonomic tool.

Frisse LM et al. (1997) International C. elegans Meeting "IDENTIFICATION OF MISMATCH REPAIR GENES IN C. ELEGANS"

Thomas WK, Lam KM, Vida JT, Frisse LM

[International C. elegans Meeting, 1997]

The MRS is critical for maintenance of genome stability and faithful genetic inheritance. Currently, Saccharomyces cerevisiae is the only eukaryotic system in which homologs of mutS and mutL have been studied thoroughly. MutS and MutL homologs of yeast MRS suppress error rates during replication and control recombination during meiosis. Deficiencies in MRS homologs of yeast display mismatch repair deficiency, microsatellite instability, and meiotic nondisjuction. We have identified four putative Caenorahbditis elegans MRS homologs by searching the C. elegans genome database using functionally characterized yeast MutS and MutL homologs as query sequences. The nematode homologs have been compared with homologs from bacteria, yeast, and other organisms in order to develop a phylogenetic hypothesis of relationships. Based on the relationships, we have identified and proposed functions for these genes. mlh-1, a homolog to yeast MLH1, and msh-2, a homolog to yeast MSH-2, may both be directly involved in mismatch repair. msh-4, a homolog to yeast MSH4, and msh-5, a homolog to yeast MSH5, are probably both involved in meitic recombination.

Sivaranjani M et al. (2016) Int J Food Microbiol "Morin inhibits biofilm production and reduces the virulence of Listeria monocytogenes - An in vitro and in vivo approach."

Ravi AV, Balamurugan K, Kamaladevi A, Sivaranjani M, Pandian SK, Gowrishankar S

[Int J Food Microbiol, 2016]

The current study explores the in vitro and in vivo antibiofilm efficacy of morin against a leading foodborne pathogen-Listeria monocytogenes (LM). Minimum inhibitory concentration (MIC) of morin against LM strains was found to be 100g/ml. The non-antibacterial effect of morin at its sub-MICs (6.25, 12.5 and 25g/ml) was determined through growth curve and XTT assay. Morin at its sub-MICs demonstrated a significant dose dependent inhibitory efficacy against LM biofilm formation which was also evidenced through light, confocal and scanning electron microscopic analyses. However, morin failed to disperse the mature biofilm of LM even at its MIC. Our data also revealed the anti-virulence efficacy of morin, as it significantly inhibited the production of hemolysin and motility of LM. Concentration-dependent susceptibility of morin treated LM cells to normal human serum was observed. In vivo studies revealed that morin extended the lifespan of LM infected Caenorhabditis elegans by about 85%. Furthermore, the non-toxic nature and in vivo anti-adherence efficacy of morin were also ascertained through C. elegans-LM infection model. Overall, the data of the current study identifies morin as a promising antibiofilm agent and its suitability to formulate protective strategies against biofilm associated infections caused by LM.

Gowrishankar S et al. (2016) Pathog Dis "Cyclic dipeptide-cyclo(L-leucyl-L-prolyl) from marine Bacillus amyloliquefaciens mitigates biofilm formation and virulence in Listeria monocytogenes."

Pandian SK, Kamaladevi A, Ravi AV, Gowrishankar S, Balamurugan K, Sivaranjani M

[Pathog Dis, 2016]

The current study was intentionally focused on cyclo(L-leucyl- L-prolyl) (CLP)-a cyclic dipeptide with myriad pharmaceutical significance, to explore its antivirulence efficacy against the predominant food-borne pathogen-Listeria monocytogenes (LM). Minimum inhibitory concentration (MIC) of CLP against LM ATCC 19111 was found to be 512 g mL(-1). CLP at sub-MICs (64,128, 256 g mL(-1)) demonstrated a profound non-bactericidal dose-dependent antibiofilm efficacy (on polystyrene and glass) against LM, which was further confirmed through confocal and scanning electron microscopic analysis (on stainless steel surface). In vitro bioassays divulged the phenomenal inhibitory efficacy of CLP towards various virulence traits of LM, specifically its overwhelming suppression of swimming and swarming motility. Data of in vivo assay using Caenorhabditis elegans signified that the plausible mechanism of CLP could be by impeding the pathogen's initial adhesion and thereby attenuating the biofilm assemblage and its associated virulence. This was further confirmed by significant decrease in exopolymeric substance, auto-aggregation, hydrophobicity index and extracellular DNA (eDNA) of the CLP treated-LM cells. Collectively, the current study unveils the antivirulence efficacy of CLP against the Gram-positive food borne-pathogen and the strain Bacillus amyloliquifaciens augurs well to be a promising probiotic in controlling infections associated with LM.

Zhao P et al. (2023) Nat Methods "Femtosecond laser microdissection for isolation of regenerating C. elegans neurons for single-cell RNA sequencing."

Jiang N, Ma KY, Chizari S, Ben-Yakar A, DuPlissis A, Martin C, Mondal S, Messing RO, Maiya R, Zhao P

[Nat Methods, 2023]

Our understanding of nerve regeneration can be enhanced by delineating its underlying molecular activities at single-neuron resolution in model organisms such as Caenorhabditis elegans. Existing cell isolation techniques cannot isolate neurons with specific regeneration phenotypes from C. elegans. We present femtosecond laser microdissection (fs-LM), a single-cell isolation method that dissects specific cells directly from living tissue by leveraging the micrometer-scale precision of fs-laser ablation. We show that fs-LM facilitates sensitive and specific gene expression profiling by single-cell RNA sequencing (scRNA-seq), while mitigating the stress-related transcriptional artifacts induced by tissue dissociation. scRNA-seq of fs-LM isolated regenerating neurons revealed transcriptional programs that are correlated with either successful or failed regeneration in wild-type and dlk-1 (0) animals, respectively. This method also allowed studying heterogeneity displayed by the same type of neuron and found gene modules with expression patterns correlated with axon regrowth rate. Our results establish fs-LM as a spatially resolved single-cell isolation method for phenotype-to-genotype mapping.

Zhao, P. et al. (2019) International Worm Meeting "Femtosecond laser microdissection isolates single C. elegans neurons for nerve regeneration RNA-seq studies."

Kreeger, L., Arur, S., ZHAO, P., Ben-Yakar, A., Trimmer, K., Messing, R., Ma, K., Martin, C., Zemelman, B., Jiang, N., Maiya, R.

[International Worm Meeting, 2019]

C. elegans has become a versatile system for studying in vivo nerve regeneration since the advent of precise laser axotomy method for severing specific axons. Through mutant and RNAi screening, a number of regeneration regulator genes have been identified. Nevertheless, their downstream effectors remain elusive. As a complementary approach, we propose to perform single-cell RNA-sequencing on regrowing neurons to capture the genome-wide dynamics underlying nerve regeneration. However, it has been technically unfeasible to isolate regrowing neurons from living C. elegans. The prevalent isolation method uses FACS to sort neurons of interest from chemo-mechanically dissociated animals, thus requires thousands of animals with synchronized nerve injury, which cannot be obtained even with state-of-the-art automated microfluidic systems. We developed a new femtosecond laser microdissection (fs-LM) method to rapidly and precisely isolate single cells directly from living tissue or organisms by leveraging femtosecond laser ablation as a high-precision cutting tool. Compared to traditional laser capture microdissection, our method provides a few crucial advantages. 1) fs-LM yields intact single cells without sample sectioning, freezing, or fixing, thus preventing sample degradation or contamination. 2) compared to the dissociation and sorting method, fs-LM induces less stress response in isolated cells. 3) fs-LM preserves the spatial and phenotypic information of the collected neurons. In addition, by correlating gene expression to the context-dependent regeneration phenotypes, it is possible to further dissect the genetic activities encoding nerve regeneration. 4) fs-LM does can isolate unlabeled cells. We isolated regrowing posterior lateral microtubule (PLM) neurons from larval 4 stage animals. Single cell RNA-sequencing on the isolated neurons identified gene expression patterns underlying axon regeneration. To demonstrate the versatility of our method, we have also dissected and sequenced single C. elegans oocytes and mammalian brain neurons.

Schieber NL et al. (2017) Methods Cell Biol "Minimal resin embedding of multicellular specimens for targeted FIB-SEM imaging."

Markert SM, Schieber NL, Stigloher C, Schwab Y, Steyer AM, Machado P

[Methods Cell Biol, 2017]

Correlative light and electron microscopy (CLEM) is a powerful tool to perform ultrastructural analysis of targeted tissues or cells. The large field of view of the light microscope (LM) enables quick and efficient surveys of the whole specimen. It is also compatible with live imaging, giving access to functional assays. CLEM protocols take advantage of the features to efficiently retrace the position of targeted sites when switching from one modality to the other. They more often rely on anatomical cues that are visible both by light and electron microscopy. We present here a simple workflow where multicellular specimens are embedded in minimal amounts of resin, exposing their surface topology that can be imaged by scanning electron microscopy (SEM). LM and SEM both benefit from a large field of view that can cover whole model organisms. As a result, targeting specific anatomic locations by focused ion beam-SEM (FIB-SEM) tomography becomes straightforward. We illustrate this application on three different model organisms, used in our laboratory: the zebrafish embryo Danio rerio, the marine worm Platynereis dumerilii, and the dauer larva of the nematode Caenorhabditis elegans. Here we focus on the experimental steps to reduce the amount of resin covering the samples and to image the specimens inside an FIB-SEM. We expect this approach to have widespread applications for volume electron microscopy on multiple model organisms.

Lucas MS et al. (2012) Methods Cell Biol "Bridging microscopes: 3D correlative light and scanning electron microscopy of complex biological structures."

Gasser P, Guenthert M, Lucas F, Lucas MS, Wepf R

[Methods Cell Biol, 2012]

The rationale of correlative light and electron microscopy (CLEM) is to collect data on different information levels--ideally from an identical area on the same sample--with the aim of combining datasets at different levels of resolution to achieve a more holistic view of the hierarchical structural organization of cells and tissues. Modern three-dimensional (3D) imaging techniques in light and electron microscopy opened up new possibilities to expand morphological studies into the third dimension at the nanometer scale and over various volume dimensions. Here, we present two alternative approaches to correlate 3D light microscopy (LM) data with scanning electron microscopy (SEM) volume data. An adapted sample preparation method based on high-pressure freezing for structure preservation, followed by freeze-substitution for multimodal en-bloc imaging or serial-section imaging is described. The advantages and potential applications are exemplarily shown on various biological samples, such as cells, individual organisms, human tissue, as well as plant tissue. The two CLEM approaches presented here are per se not mutually exclusive, but have their distinct advantages. Confocal laser scanning microscopy (CLSM) and focused ion beam-SEM (FIB-SEM) is most suitable for targeted 3D correlation of small volumes, whereas serial-section LM and SEM imaging has its strength in large-area or -volume screening and correlation. The second method can be combined with immunocytochemical methods. Both methods, however, have the potential to extract statistically relevant data of structural details for systems biology.

Kiontke K et al. (2000) European Worm Meeting ""Saltational evolution" of phasmid position in nematodes"

Sudhaus W, Kiontke K, Fitch DHA

[European Worm Meeting, 2000]

Phasmids are structures in the tail region of secernentean nematodes. The two large groups within Nematoda, Secernentea and "Adenophorea", are distinguished by this character: "Adenophorea" lack phasmids. Phasmids are similarly built in C. elegans(Rhabditidae) Tylenchidae and Filariidae. They consist of 1-2 socket cells that contact the body epidermis, a glandular sheath cell, and one or two sensory processes projecting into a receptor cavity within the sheath cell. Through a pore in the socket cell, these recessed processes are exposed to the exterior. Phasmids are generally described for females of most secernentean species. However, especially in Rhabditidae, phasmids in males have rarely been reported. This is due to the fact that in males phasmids are easily confused with rays if they are integrated into the velum (Fitch & Emmons, 1995). With SEM and interference contrast LM, the pore in the phasmid socket cell is clearly visible, whereas in rays either one sensory process protrudes through an opening in the structural cell or this opening is very small. We studied 53 species of Rhabditidae including Heterorhabditis as well as Diplogastrina, Panagrolaimidae, Cephalobidae, Brevibuccidae, Myolaimus, Steinernema, and Strongylida with LM and SEM, and scanned the literature on animal parasitic Secernentea. Phasmids are present in males of all species. The rhabditid ancestor had 9 pairs of rays and one pair of phasmids instead of 10 pairs as rays as was previously assumed. Two alternative positions of the phasmids relative to the rays could be distinguished: an anterior position with 3-4 rays posterior to the phasmid, and a posterior position with all rays anterior to the phasmid as in C. elegans. There are never more than 4 rays posterior to the phasmid. Phasmids are anterior in Cephalobidae and Diplogastrina and posterior in Panagrolaimidae, in Steinernema, and in Strongylidae. Within Rhabditidae both character states occur. We mapped the phasmid position on a cladogram based on small subunit rDNA (Fitch et al. unpublished) and found that multiple changes between anterior and posterior phasmid position must have occurred during evolution. This could be explained in terms of the development of phasmid socket cells and the posterior three rays, which are all derived from the same blast cell (T) in the L1 larva. In C. elegans, the phasmid socket cells are descendants of the posterior daughter of the T cell. The polarity of the first division of the T cell might be reversed in species with anterior phasmids, such that the phasmid socket cells are now descendants of its anterior daughter (Fitch, 1997; Kiontke & Sudhaus in press). We have begun to test this hypothesis. References: Fitch, D.H.A. & Emmons S. (1995) Dev. Biol. 170: 564-582 Fitch, D.H.A. (1997) Syst. Biol. 56: 145-179. Kiontke, K. & Sudhaus, W. (in Press) J. Nemat. Morph. Syst.