Calhoun, W.A. et al. (2019) International Worm Meeting "An open-top light sheet microscope with remote focusing for imaging neural activity of C. elegans in microfluidics."

Calhoun, W.A., Lu, H.

[International Worm Meeting, 2019]

Whole brain imaging of C. elegans holds incredible potential for investigating how nervous systems integrate sensory input and drive behavior. This is particularly true with the application of microfluidics to precisely manipulate worms' sensory environment. However, collecting high-quality volumetric images with sufficient speed to track individual neurons as a worm moves and deforms tests the limits of optical microscopy. Light sheet microscopy has proven to have several advantages for high-speed volumetric imaging, including low light exposure and high collection efficiency, which make it ideal for imaging small, transparent specimens. However, the geometry of conventional light sheet microscopes requires that the sample be suspended in the small volume between the separate illumination and detection objectives. This renders these microscopes incompatible with microfluidics, agarose pads, or any other planar sample mounting which would be physically blocked by the objectives. We present an open-top light sheet microscope capable of fast volumetric imaging that is compatible with planar microfluidics and demonstrate its utility for recording neural activity in C. elegans. This design uses remote focusing with an electrically tunable lens to rapidly reposition the image plane without physically moving the detection objective, enabling acquisition at camera-limited volume rates. We show that multi-channel fluorescence imaging at 10+ volumes/second with light sheet microscopy improves whole brain calcium imaging and reduces motion artifacts. We also demonstrate its ability to record fine anatomical features during mechanical stimulation through microfluidics.

Katya V Melnik-Martinez et al. (2004) West Coast Worm Meeting "Claudin-like Caenorhabditis Protein-3 a putative Voltage Gated Calcium Channel gamma subunit"

Stefan Herlitze, Katya V Melnik-Martinez, William R Scahfer

[West Coast Worm Meeting, 2004]

Voltage gated calcium channels (VGCCs) mediate Ca2+ entry into cells in response to membrane depolarizations (Catterall, W.A., 2000). VGCCs are composed of different subunits: a pore forming a1 subunit, a transmembrane disulfide-linked complex of a2 and d subunits, an intracellular b subunit and a transmembrane g subunit. In order to study the role of the g subunit in C. elegans we have identified in Worm Base a putative g subunit, Claudin-like Caenoharbditis protein 3(CLC-3). Using a clc-3 promoter::GFP construct we show that CLC-3 is expressed in the nerve ring, ventral cord and phasmid neurons. Preliminary analysis of a clc-3 C. elegans deletion mutant reveals locomotion defects consistent with abnormal motorneuron function. Imaging data adressing the effects of the clc-3 deletion on neuronal calcium transients in vitro and in vivo will be presented

B Podbilewicz et al. (1999) International C. elegans Meeting "Cold sensitive fusion: kinetic dissection of cell fusion during morphogenesis in N2 and duf-1"

T Gattegno, B Podbilewicz, Y Rabin, M Glickman

[International C. elegans Meeting, 1999]

Cell fusion is an essential process in development conserved throughout evolution. We have analysed the sequence of epithelial cell fusions in C. elegans [1] and have isolated mutations that disrupt cell fusion in the embryo. To elucidate the molecular mechanisms of cell fusion in situ we arrested or slowed down this process by lowering the temperature. We used confocal microscopy and a custom-made cooling stage to record 4D-timelapse movies of developing embryos expressing MH27-GFP fusion protein in the adherens junctions[2] at different temperatures. At temperatures below 4 o C, comma stage embryos did not elongate and epithelial fusions did not occur. However, at 5 o C embryos elongated to 2-fold at a rate of about 20-fold slower than that at 20 o C and dorsal cell fusions did not occur. Moreover, this treatment phenocopies the arrest of duf-1(zu316cs) embryos at 15 o C[3]. The fusion rate measured from timelapse recordings as disappearance of MH27-GFP staining varied from 50 nm/min at 6 o C to 1000 nm/min at 27.5 o C. Thus, we show that the cell fusion rate is strongly dependent on temperature. Using a combination of kinetic and genetic analyses we have initiated the dissection of the multi-step process involving the opening and expansion of fusion pores necessary for morphogenesis and the formation of organs in C. elegans and other organisms. We propose that cell fusion in C. elegans can be dissected into at least three steps: (1) transient hemifusion; (2) rearrangements of two lipid bilayers into one (fusion pore formation arrested at 5 o C); and (3) expansion of fusion pore (estimated fusion rates at 6 o C). Using Arrhenius plots for N2 and duf-1 we can get information on the mechanisms of pore expansion during cell fusion in vivo. The energy of activation can be estimated and compared between wild-type and mutant. [1] Podbilewicz B. and White J.G. (1994) Dev. Biol. 161, 408-424. [2] Mohler, W.A et al (1998) Curr. Biol. 8, 1087-1090. [3] Gattegno T. (1999) IWM Abstract.

T Gattegno et al. (1999) International C. elegans Meeting "duf-1(zu316cs) - Dorsal UnFused"

B Podbilewicz, T Gattegno

[International C. elegans Meeting, 1999]

During embryonic development of C.elegans , hypodermal cells undergo programmed cell fusion to create multi-nucleated cells. Though the temporal and spatial patterns of the fusion events in the wild type have been characterized the molecular mechanism that induces cell fusion is unclear(1). Analysis of mutants affected in the fusion process provides an opportunity to clone the genes involved. duf-1(zu316cs) has fewer fusion events in the dorsal hypodermis compared to wild type(2) and it was isolated during a screen for elongation defects(3). Embryonic elongation occurs simultaneously with a series of fusion events at the hypodermis. We have characterized zu316cs as a zygotic lethal cold sensitive mutation and mapped it to the left arm of chromosome X, between -15.81 and -17.26 in the genetic map. The arrest phenotype was characterized using Nomarski optics; arrested embryos continue to move and have a twisted tail(2). Fusion analysis was done using the monoclonal antibody MH27 that recognizes the apical boundaries of hypodermal cells(1); a strain expressing MH27-GFP fusion protein(4) and carrying zu316cs enables efficient characterization of living embryos. MAbs indicative for muscles, intestine, pharynx, glial-like cells and hypodermis were used to characterize zu316cs . Staining patterns were fundamentally normal implying that zu316cs is specifically affected in the hypodermal fusion process. We further analyzed the number and identity of the unfused cells by counting junctions in arrested embryos (7.6+/-3.2; n=61) in comparison to wild type embryos at similar elongation stages (1.5+/-0.5; n=15). zu316cs arrest phenotype at the restrictive temperature can be artificially created by growing wild type embryos at 5degC(5). These results suggest conditional relationship between elongation and fusion; elongation does not proceed beyond 2 fold when hypodermal fusion is affected. Phenotypic and molecular characterization of duf-1 will contribute to a better understanding of the association between elongation and fusion processes. [1] Podbilewicz B. and White J.G. (1994) Dev. Biol. 161, 408-424. [2] Gattegno T. and Podbilewicz B. (1997) IWM Abstract, p.546. [3] Costa M. and Priess J. (1995) IWM Abstract, p.165. [4] Mohler W.A., et al. (1998) Curr Biol. 8, 1087-1090. [5] Podbilewicz B. (1999) IWM Abstract.