Ames, K. et al. (2013) International Worm Meeting "Role of autophagy genes in C.elegans germline proliferation."

Ames, K., Melendez, A.

[International Worm Meeting, 2013]

Autophagy is a conserved pathway characterized by the formation of double-membrane vesicles to degrade cellular material and maintain cellular homeostasis. BEC-1, the C. elegans ortholog of mammalian Beclin1 and yeast Atg6/Vps30, is essential in the vesicle nucleation step of autophagosome formation. In C. elegans, we have demonstrated that BEC-1 is crucial for various aspects of development. Importantly, beclin1 is a haploinsufficient tumor suppressor gene in mice and the beclin1 gene is monoallelically deleted in various human cancers. However, the role of autophagy in tumor growth is not well understood. To better understand how bec-1 regulates cell proliferation, we used the C. elegans germline model. Proliferation of the germline is determined by GLP-1/Notch signaling. Mutations that increase glp-1 signaling result in an overproliferation phenotype, whereas, reduced glp-1 signaling results in the lack of germline proliferation and premature meiotic entry. We found that genetically reducing the function of bec-1, atg-16.2 or atg-18 results in decreased number of germline mitotic cells, suggesting that autophagy contributes to the proliferation and/or maintenance of the mitotic pool of germ cells. Moreover, compromising autophagy function in glp-1(gf) mutants by genetic removal of bec-1, atg-16.2 or atg-18, significantly decreased mitotic cell proliferation of glp-1(gf) mutants, as did RNAi against autophagy genes. We conclude that autophagy genes play a role in mediating the proliferation of undifferentiated cells in the glp-1(gf) tumor model. We found that the observed effect of reduced autophagy on proliferation is neither due to an increase in cell death nor due to a premature exit from mitosis. Moreover, is not due to an initial lack of proliferation during development. Thus, we hypothesize that the decrease in autophagy gene function may result in defects in cell cycle progression. Future experiments will test this hypothesis. We expect that our studies will help better understand the role of bec-1 and autophagy in the germline proliferation and identify the molecular mechanisms by which autophagy genes modulate proliferation and/or maintenance of the germline stem cell population.

Ames K et al. (2017) Curr Biol "A Non-Cell-Autonomous Role of BEC-1/BECN1/Beclin1 in Coordinating Cell-Cycle Progression and Stem Cell Proliferation during Germline Development."

Konta M, Ames K, Bulow HE, Shechter G, Starikov L, Wong S, Da Cunha DS, Lin F, Melendez A, Gonzalez B

[Curr Biol, 2017]

The decision of stem cells to proliferate and differentiate is finely controlled. The Caenorhabditis elegans germline provides a tractable system for studying the mechanisms that control stem cell proliferation and homeostasis [1-4]. Autophagy is a conserved cellular recycling process crucial for cellular homeostasis in many different contexts [5], but its function in germline stem cell proliferation remains poorly understood. Here, we describe a function for autophagy in germline stem cell proliferation. We found that autophagy genes such as bec-1/BECN1/Beclin1, atg-16.2/ATG16L, atg-18/WIPI1/2, and atg-7/ATG7 are required for the late larval expansion of germline stem cell progenitors in the C.elegans gonad. We further show that BEC-1/BECN1/Beclin1 acts independently of the GLP-1/Notch or DAF-7/TGF- pathways but together with the DAF-2/insulin IGF-1 receptor (IIR) signaling pathway to promote germline stem cell proliferation. Similar to DAF-2/IIR, BEC-1/BECN1/Beclin1, ATG-18/WIPI1/2, and ATG-16.2/ATG16L all promotecell-cycle progression and are negatively regulated by the phosphatase and tensin homolog DAF-18/PTEN.However, whereas BEC-1/BECN1/Beclin1 acts through the transcriptional regulator SKN-1/Nrf1, ATG-18/WIPI1/2 and ATG-16.2/ATG16L exert their function through the DAF-16/FOXO transcription factor. In contrast, ATG-7 functions in concert with the DAF-7/TGF- pathway to promote germline proliferation and is not required for cell-cycle progression. Finally, we report that BEC-1/BECN1/Beclin1 functions non-cell-autonomously to facilitate cell-cycle progression and stem cell proliferation. Our findings demonstrate a novel non-autonomous role for BEC-1/BECN1/Beclin1 in the control of stem cell proliferation and cell-cycle progression, which may have implications for the understanding and development of therapies against malignant cell growth in the future.

Wilcox K (2009) Sci Am "Free radical shift."

Wilcox K

[Sci Am, 2009]

Strange K (2000) Am J Physiol Cell Physiol "Model organisms: comparative physiology or just physiology?"

Strange K

[Am J Physiol Cell Physiol, 2000]

A new field referred to as "functional genomics" (or, from a physiologist's perspective, "physiological genomics") has emerged in the wake of genome sequencing. Functional genomics is defined as the assignment of function to identified genes and, importantly, the elucidation of the organization, integration, and control of networks of proteins that give rise to specific biological processes. It is widely recognized that the significant challenges posed by attempts to define the genetic basis of physiology necessitate the study of experimentally more manipulable "model organisms".

Strange K (2005) Am J Physiol Cell Physiol "The end of "naive reductionism": rise of systems biology or renaissance of physiology?"

Strange K

[Am J Physiol Cell Physiol, 2005]

Systems biology is an emerging discipline focused on tackling the enormous intellectual and technical challenges associated with translating genome sequence into a comprehensive understanding of how organisms are built and run. Physiology and systems biology share the goal of understanding the integrated function of complex, multicomponent biological systems ranging from interacting proteins that carry out specific tasks to whole organisms. Despite this common ground, physiology as an academic discipline runs the real risk of fading into the background and being superseded organizationally and administratively by systems biology. My goal in this article is to discuss briefly the cornerstones of modern systems biology, specifically functional genomics, nonmammalian model organisms and computational biology, and to emphasize the need to embrace them as essential components of 21st-century physiology departments and research and

Darge K et al. (1994) Am J Trop Med Hyg "Evaluation of ultrasonography for the detection of drug-induced changes in onchocercal nodules."

Darge K, Buettner DW, Nelle M, Awadzi K, Troeger J, Leichsenring M, Engelke C

[Am J Trop Med Hyg, 1994]

Clinical trials of macrofilaricidal drugs against Onchocerca volvulus are impeded due to the lack of means for assessing in vivo drug-induced changes in the onchocercomas. The application of ultrasonography in the sequential monitoring of morphologic alterations of onchocercal nodules after six weeks of suramin therapy was evaluated in 20 male patients from Ghana with a total of 64 nodule sites. After each follow-up session, a number of onchocercal nodules were extirpated so that by the end of one year, all nodules had been removed for histologic examination. The sonomorphologic changes observed and their time of appearance correlated well with the histologic findings of the onchocercomas. Eighty-three percent of the onchocercal nodules became hyperechogenic and 22% developed echo-free areas at the end of the follow-up period. Absence of the lateral acoustic shadow increased by more than 30% and the lack of differentiation of the worm center from the capsule and the nodule from its surrounding tissue increased by the end of one-year posttreatment to 100% and 91%, respectively. A mean reduction of nodule size of 27% was also documented. The histologic studies revealed that the proportion of the dead female worms increased from 17% at the end of the suramin therapy to 48% six months later and reached 61% at one year. It is concluded that ultrasonographic monitoring of onchocercomas can provide essential information on drug effects and facilitate clinical trials of macrofilaricidal drugs, limiting histologic evaluation to a few objectively selected onchocercomas.

Platzer K et al. (2018) Am J Hum Genet "De Novo Variants in MAPK8IP3 Cause Intellectual Disability with Variable Brain Anomalies."

Stegmann APA, Bonati MT, Panis B, Smith-Hicks C, Lemke JR, Pepler A, Wilson C, Iascone M, McWalter K, Brasington C, Allen W, Di Donato N, Platzer K, Ramos L, Edwards SL, Jamra R, Gamble CN, Mandel H, Stobe P, Mahida S, Marquardt T, Demmer LA, Miller KG, Falik-Zaccai T, Pinz H, Hellenbroich Y, Sticht H, Kok F, Cho MT, Stumpel CTRM, Shinde DN, Angione KM

[Am J Hum Genet, 2018]

Using exome sequencing, we have identified de novo variants in MAPK8IP3 in 13 unrelated individuals presenting with an overlapping phenotype of mild to severe intellectual disability. The de novo variants comprise six missense variants, three of which are recurrent, and three truncating variants. Brain anomalies such as perisylvian polymicrogyria, cerebral or cerebellar atrophy, and hypoplasia of the corpus callosum were consistent among individuals harboring recurrent de novo missense variants. MAPK8IP3 has been shown to be involved in the retrograde axonal-transport machinery, but many of its specific functions are yet to be elucidated. Using the CRISPR-Cas9 system to target six conserved amino acid positions in Caenorhabditis elegans, we found that two of the six investigated human alterations led to a significantly elevated density of axonal lysosomes, and five variants were associated with adverse locomotion. Reverse-engineering normalized the observed adverse effects back to wild-type levels. Combining genetic, phenotypic, and functional findings, as well as the significant enrichment of de novo variants in MAPK8IP3 within our total cohort of 27,232 individuals who underwent exome sequencing, we implicate de novo variants in MAPK8IP3 as a cause of a neurodevelopmental disorder with intellectual disability and variable brain anomalies.

Balamurugan K et al. (2007) Am J Physiol Cell Physiol "Cloning and functional characterization of a folate transporter from the nematode Caenorhabditis elegans."

Said HM, Balamurugan K, Sze JY, Ashokkumar B, Moussaif M

[Am J Physiol Cell Physiol, 2007]

Two putative orthologs to the human reduced folate carrier (hRFC) folt-1 and folt-2 that share a 40% and 31% identity, respectively, with the hRFC sequence have been identified in the C. elegans genome. Functional characterization of the open reading frame of putative folt-1 and folt-2 showed the folt-1 to be a specific folate transporter. Transport of folate by folt-1 expressed in a heterologous expression system showed an acidic pH-dependence, saturability (apparent Km of 1.23 +/- 0.18 microM), similar degree of inhibition by reduced and substituted folate derivatives, sensitivity to the anti-inflammatory drug sulfasalazine (apparent Ki of 0.13 mM), and inhibition by the anion transport inhibitors, e.g., 4,4''-diisothio-cyanatostilbene-2,2''-disulphonic acid (DIDS). Knocking down or knocking out the folt-1 gene led to a significant inhibition in folate uptake by intact living C. elegans. We also cloned the 5''-regulatory region of folt-1 gene, and confirmed promoter activity of the construct in vivo in living C. elegans. Using the transcriptional fusion construct (i.e., folt-1::GFP), the expression pattern of folt-1 in different tissues of living animal was found to be highest in the pharynx and intestine. Also, folt-1::GFP expression was developmentally and adaptively regulated in vivo. These studies demonstrate for the first time the existence of a specialized folate uptake system in C. elegans that has similar characteristics to that of folate uptake process of the human intestine. Thus, C. elegan provides a genetically tractable model that can be used for integrative aspects of the folate uptake process in the context of the whole animal level. Key words: C. elegans, integrative transport physiology, folate transport, transport regulation.

Burkewitz K et al. (2011) Am J Physiol Cell Physiol "Hypertonic stress induces rapid and widespread protein damage in C. elegans."

Strange K, Choe K, Burkewitz K

[Am J Physiol Cell Physiol, 2011]

Proteostasis is defined as the homeostatic mechanisms that maintain the function of all cytoplasmic proteins. We recently demonstrated that the capacity of the proteostasis network is a critical factor that defines the limits of cellular and organismal survival in hypertonic environments. The current studies were performed to determine the extent of protein damage induced by cellular water loss. Using worm strains expressing fluorescently tagged foreign and endogenous proteins and proteins with temperature-sensitive point mutations, we demonstrate that hypertonic stress causes aggregation and misfolding of diverse proteins in multiple cell types. Protein damage is rapid. Aggregation of a polyglutamine yellow fluorescent protein reporter is observable with <1 h of hypertonic stress, and aggregate volume doubles approximately every 10 min. Aggregate formation is irreversible and occurs after as little as 10 min of exposure to hypertonic conditions. To determine whether endogenous proteins are aggregated by hypertonic stress, we quantified the relative amount of total cellular protein present in detergent-insoluble extracts. Exposure for 4 h to 400 mM or 500 mM NaCl induced a 55-120% increase in endogenous protein aggregation. Inhibition of insulin signaling or acclimation to mild hypertonic stress increased survival under extreme hypertonic conditions and prevented aggregation of endogenous proteins. Our results demonstrate that hypertonic stress causes widespread and dramatic protein damage and that cells have a significant capacity to remodel the network of proteins that function to maintain proteostasis. These findings have important implications for understanding how cells cope with hypertonic stress and other protein-damaging stressors.

Chun-Yi Huang et al. (2007) International Worm Meeting "eif-3.K is a pro-apoptotic gene in C. elegans."

Jia-Yun Chen, Yi-Chun Wu, Chun-Yi Huang

[International Worm Meeting, 2007]

Programmed cell death (or apoptosis) is an important feature of C. elegans development. Previous studies have identified pro-apoptotic genes egl-1, ced-3 and ced-4 and anti-apoptotic genes ced-9 and icd-1 that control programmed cell death.. We have identified and characterized a novel pro-apoptotic gene eif-3.K. Loss-of-function by mutation or RNAi inactivation in eif-3.K resulted in a decrease of cell corpses, whereas heatshock-induced over-expression of eif-3.K weakly but significantly increased cell corpses. Interestingly, the eif-3.K mutation partially suppressed ectopic cell deaths caused by over-expression of egl-1 or ced-4. This result suggests that eif-3.K may act downstream of or in parallel to egl-1 and ced-4 in the programmed cell death pathway. Using a cell-specific promoter to express eif-3.k in touch neurons, we showed that eif-3.K likely promoted cell death in a cell-autonomous manner. To further explore EIF-3.K function, we generated antibodies against bacterially expressed EIF-3.K protein. We found that EIF-3.K was ubiquitously expressed during embryogenesis and localized to the cytoplasm. As human eif-3.K can functionally substitute C. elegans eif-3.K in an eif-3.K mutant, the function of eif-3.K in apoptosis is likely conserved in evolution.