Dana Byrd et al. (2008) Development & Evolution Meeting "Subcellular Architecture of the Germline Stem Cell Niche"

Karla Knobel, Judith Kimble, Katharyn Schmitt, Dana Byrd, Audra Amasino

[Development & Evolution Meeting, 2008]

A stem cell's decision between self-renewal and differentiation is governed by extrinsic stimuli from the surrounding microenvironment, or niche, as well as by intrinsic cues. In C. elegans, the distal tip cell (DTC) provides the niche for germline stem cells and maintains a "mitotic region" by Notch signaling (e.g. Kimble and Crittenden 2007). DTC number and dosage of the LAG-2 DSL ligand, which is expressed by the DTC, can affect the number of germ cells in the mitotic region (Dyan Vogel and Myon-Hee Lee). To investigate DTC niche function, we made a panel of fluorescent protein markers to visualize its subcellular structure in vivo. These include markers for the nuclear envelope, endoplasmic reticulum, Golgi, endosomes, and plasma membrane. Using membrane markers, we found that contact between the DTC niche and germ cells in the mitotic region is extensive. The DTC extends membrane processes that surround adjacent germ cells. The intimate contact between the niche and germ cells may provide a mechanism to physically anchor the distal-most germ cells within the niche and/or provide more localized signaling to maintain the germline stem cells. With a combination of DTC, sheath cell, and germ cell markers, we are examining the relationship between the extent of niche contact and the region of cells with stem cell potential. We are also using tissue-specific RNAi to test which genes are required in the DTC for regulation of germline stem cells.

Knapp-Wilson, A. et al. (2017) International Worm Meeting "Investigating Complex I activity and mitochondrial protein import in the multi-cellular model organism C. elegans."

Collinson, I., Knapp-Wilson, A., Kuwabara, P.

[International Worm Meeting, 2017]

Mitochondria are small cytoplasmic organelles essential for meeting the majority of cellular energy demands via the oxidative phosphorylation (OXPHOS) pathway. [1, 2]. They feature a double membrane that separates the organelle into four distinct compartments [3]. The vast majority of the work on mitochondria has been carried out in Saccharomyces cerevisiae, a single-celled genetically tractable eukaryote [4]. Although biochemically competent; the model has limitations in its applicability to humans. The mitochondrial structure, metabolism and bioenergetics of the nematode are very similar to the mammalian counterpart [5], this conservation of mitochondrial function and the technological and anatomical advantages offered by the nematode make it an excellent model organism to explore mitochondrial function and activity. [1, 6] My project is multidisciplinary, using a broad range of techniques including whole animal physiology, biochemical and structural analysis, and molecular dynamic simulations to gain a better understanding of the mitochondrial respiratory machinery, and the impact dysfunction of this machinery can have. Mitochondrial dysfunction leads to many serious human diseases and a better understanding of the mechanisms involved is essential in future medical applications. [1] Maglioni, S. & Ventura, N., 2016. C. elegans as a model organism for human mitochondrial associated disorders. Mitochondrion, 30, pp.117-125. [2] Lemire, B. Mitochondrial genetics (September 14, 2005), WormBook, ed. The C. elegans Research Community, WormBook, doi/10.1895/wormbook.1.25.1, [3] McBride et al, 2006. Mitochondria: more than just a powerhouse., 16(14), pp.R551-60 [4] Tokatlidis, K., et al. (2000). "Membrane protein import in yeast mitochondria." Biochem Soc Trans 28(4): [5] Murfitt, R.R., Vogel, K. & Sanadi, D.R., 1976. Characterization of the mitochondria of the free-living nematode, Caenorhabditis elegans. Comparative biochemistry and physiology. B, Comparative biochemistry, 53(4), [6] Tsang, W.Y. & Lemire, B.D., 2003. The role of mitochondria in the life of the nematode, Caenorhabditis elegans. Biochimica et biophysica acta, 1638(2)

Martina Machackova et al. (2007) International Worm Meeting "Functional analysis of NHR-25 in C. elegans male tail differentiation."

Marek JINDRA, Martina Machackova, Masako ASAHINA

[International Worm Meeting, 2007]

A nuclear receptor NHR-25 (a homolog of the Ftz-F1 and SF-1 proteins) functions in developmental processes that involve cell-cell contact, cell motility and asymmetric cell division. The stem epidermal seam cells undergo dynamic cell shape changes and the cell-cell interaction is required for their daughters to properly differentiate. We and others have previously shown that NHR-25 plays a critical role in this epidermal differentiation as seam cell contact is perturbed and extra seam cells arise when NHR-25 function is compromised (Chen et al. 2004, Silhankova et al. 2005). Here, we have generated chromosomally integrated transgenic worms that carry nhr-25 RNAi construct driven by a seam cell specific promoter. As expected the seam cell differentiation and number was altered. The most frequently affected structure in the transgenic worms was the male tail. In the normal male tail, the lineages of the V5, V6 and T seam cells form nine sensory finger-like rays embedded in a cuticle fan and each ray has a distinct set of unique morphogenetic and molecular features. Homozygous nhr-25(RNAi) worms exhibited severe Mab (male abnormal) phenotype such as partial tail tip retraction, shrunk fan, ray absence or ray fusion, and almost all rays were affected randomly. Animals heterozygous for nhr-25(RNAi) showed less severe yet more specific anomalies: absence or displacement of ray 1, absence of ray 2, ray 1-2 or 3-4 fusions, or formation of a tenth ectopic ray. Our data therefore strongly suggest that NHR-25 is required for proper male tail differentiation. Male tail morphogenesis is known to involve several signaling pathways, thus opening an opportunity to further explore the regulatory circuitry of the nuclear receptor signaling. Crosstalk between nuclear receptors and signaling pathways is a great interest as the interaction between NHR-25 and Wnt/<font face=symbol>b</font>-catenin singling have been recently demonstrated in C. elegans gonad (Asahina et al. 2006). This work is supported by grants 204/07/0948 and 524/03/H133 from Czech Science Foundation, 2B06129 from the Czech Ministry of Education, and 50/2006/P-BF from GAJU.

Lesley Emtage et al. (2001) International C. elegans Meeting "Making the Matrix: MEC-1, a Multifunctional Extracellular Protein"

Lesley Emtage, Guoqiang Gu, Marty Chalfie

[International C. elegans Meeting, 2001]

In electron micrographs, the C. elegans touch receptor neurons have two distinguishing characteristics: a bundle of 15-protofilament microtubules and extensive extracellular matrix. In the current molecular model for touch sensitivity both of these feature are thought to provide tethering points for the mechanosensory channel. mec-1 is needed for the elaboration of the extracellular matrix. The mec-1 gene was first identified by mutations that cause touch insensitivity. In the initial characterization of mec-1 mutants (Chalfie and Sulston, 1981), loss of mec-1 was found to affect the touch cells in two ways: it caused the loss of most of the extracellular matrix and it prevented the attachment of touch cell processes near the cuticle and the engulfment of lateral touch cell processes by the surrounding hypodermis that normally occurs during development. These observations suggested that the attachment of the touch cells was important for their function. Since him-4 mutations prevent the attachment and engulfment without affecting touch sensitivity (Vogel and Hedgecock, 2001), attachment, per se, is not essential for touch sensitivity. We have now cloned mec-1 and find that the touch sensitivity and attachment functions map to different portions of the molecule. mec-1 encodes a 1999 amino acid polypeptide with an N-terminal signal sequence followed by a Kunitz-type domain, two EGF domains, 14 additional Kunitz-type domains, and a C-terminus of 160 amino acids. Nine of the Kunitz domains form a large cluster just prior to the C-terminus. The Kunitz and EGF domains are likely to be protein interaction domains. The only other protein with both of these domains is MEC-9, another extracellular protein needed for touch sensitivity, although a substantial class of C. elegans proteins have Kunitz domains interspersed with other extracellular matrix motifs. A mec-1::gfp fusion is expressed in the touch cells, several other lateral neurons, and the intestinal muscles. We have characterized eleven mec-1 alleles: eight are nonsense mutations or mutations at splice junctions that would lead to premature termination, two are missense mutations affecting cysteines in the last Kunitz domain, and one is a Tc1 insertion 100 or so amino acids from the C terminus (and after the EGF and Kunitz domains). The last three mutations and two of the nonsense mutations causing termination in the next to last Kunitz domain result in touch insensitive animals whose touch cells are normally engulfed. By looking at the phenotype caused by other nonsense mutations in a smg-5 background, which presumably allows for normal production of truncated proteins, we have found two additional nonsense mutations that permit normal engulfment of the touch cell processes. These mutations suggest that engulfment requires the N-terminal region through the sixth Kunitz domains, but not the large C-terminal cluster of Kunitz domains. Thus, the MEC-1 protein has a C-terminal region that is essential for mechanosensory function but not for attachment and engulfment of the touch cell processes.

Elliott J. Hagedorn et al. (2007) International Worm Meeting "Time-lapse visualization of anchor cell invasion in C. elegans."

David R. Sherwood, Elliott J. Hagedorn

[International Worm Meeting, 2007]

A lack of in vivo models has hampered insight into the mechanisms driving cell-invasive behavior. The behavior of a single uterine cell in Caenorhabditis elegans called the anchor cell (AC) provides such an in vivo model, allowing for easy visualization and genetic manipulation. During normal development in the C. elegans hermaphrodite, the AC invades through the underlying juxtaposed uterine and ventral epidermal basement membranes (BM) to establish the initial uterine-vulval contact. In addition to the genetic tractability and ease of visualization, the consistency of this event, occurring at the same time in every wild-type hermaphrodite, makes for a powerful model of cell invasion. We have established an in vivo system to visualize anchor cell invasion as it occurs in real time using a laminin-GFP fusion (LAM-1::GFP, Kao and Wadsworth 2006, Dev Biol. 290(1): 211-219) or a hemicentin-GFP fusion (GFP-hemicentin, Vogel and Hedgecock 2001, Development 128(6): 883-894), both markers of the BM, in combination with a PH::mCherry (PH domain from PLC?, a gift from Anjon Audhya) driven by an AC-specific promoter, labeling the invasive membrane (Sherwood et al. 2005, Cell 121(6): 951-962). To investigate the real-time dynamics of invasion, we have begun to characterize AC invasion in wild-type animals. Prior to invasion there is a deposition of GFP-hemicentin and an apparent increase of LAM-1::GFP in the uterine BM directly below the AC, suggesting that the AC deposits or recruits additional BM components, prior to migration through it. Invasion appears to initiate with a single filopod extending through a tiny hole in the BM. Once this filopodial process reaches through the BM, possibly contacting vulval cells below, it begins to widen, often fanning out on the ventral side of the BM. This widening of the AC extension seems to be coincident with widening of the hole in the BM, as visualized by LAM-1::GFP. The initial characterization of wild-type invasion has brought about several questions. 1) How is the initial filopod generated, and how does it penetrate through the BM? 2) What dictates the precise location of the initial protrusive filopod? 3) What happens to the BM under the AC as the filopod widens? 4) Do other BM components similarly increase under the AC, how are they recruited there, and what function does their increase serve? It is our hope that through a detailed cell-biological analysis, in combination with genetic screens and mutant analysis, that we will gain insight into these questions and lead to a better understanding of the mechanisms that endow invasive cells with the ability to traverse basement membranes.