The Caenorhabditis elegans anchor cell transcriptome: ribosome biogenesis drives cell invasion through basement membrane.

Cell invasion through basement membrane (BM) barriers is important in development, immune function and cancer progression. As invasion through BM is often stochastic, capturing gene expression profiles of actively invading cells in vivo remains elusive. Using the stereotyped timing of Caenorhabditis elegans anchor cell (AC) invasion, we generated an AC transcriptome during BM breaching. Through a focused RNAi screen of transcriptionally enriched genes, we identified new invasion regulators, including translationally controlled tumor protein (TCTP). We also discovered gene enrichment of ribosomal proteins. AC-specific RNAi, endogenous ribosome labeling and ribosome biogenesis analysis revealed that a burst of ribosome production occurs shortly after AC specification, which drives the translation of proteins mediating BM removal. Ribosomes also enrich near the AC endoplasmic reticulum (ER) Sec61 translocon and the endomembrane system expands before invasion. We show that AC invasion is sensitive to ER stress, indicating a heightened requirement for translation of ER-trafficked proteins. These studies reveal key roles for ribosome biogenesis and endomembrane expansion in cell invasion through BM and establish the AC transcriptome as a resource to identify mechanisms underlying BM transmigration.

Chronic high-sugar diet in adulthood protects Caenorhabditis elegans from 6-OHDA-induced dopaminergic neurodegeneration.

BACKGROUND: Diets high in saturated fat and sugar, termed "Western diets," have been associated with several negative health outcomes, including increased risk for neurodegenerative disease. Parkinson's disease (PD) is the second most prevalent neurodegenerative disease and is characterized by the progressive death of dopaminergic neurons in the brain. We build upon previous work characterizing the impact of high-sugar diets in Caenorhabditis elegans to mechanistically evaluate the relationship between high-sugar diets and dopaminergic neurodegeneration. RESULTS: Adult high-glucose and high-fructose diets, or exposure from day 1 to 5 of adulthood, led to increased lipid content, shorter lifespan, and decreased reproduction. However, in contrast to previous reports, we found that adult chronic high-glucose and high-fructose diets did not induce dopaminergic neurodegeneration alone and were protective from 6-hydroxydopamine (6-OHDA) induced degeneration. Neither sugar altered baseline electron transport chain function and both increased vulnerability to organism-wide ATP depletion when the electron transport chain was inhibited, arguing against energetic rescue as a basis for neuroprotection. The induction of oxidative stress by 6-OHDA is hypothesized to contribute to its pathology, and high-sugar diets prevented this increase in the soma of the dopaminergic neurons. However, we did not find increased expression of antioxidant enzymes or glutathione levels. Instead, we found evidence suggesting downregulation of the dopamine reuptake transporter dat-1 that could result in decreased 6-OHDA uptake. CONCLUSIONS: Our work uncovers a neuroprotective role for high-sugar diets, despite concomitant decreases in lifespan and reproduction. Our results support the broader finding that ATP depletion alone is insufficient to induce dopaminergic neurodegeneration, whereas increased neuronal oxidative stress may drive degeneration. Finally, our work highlights the importance of evaluating lifestyle by toxicant interactions.

Forces drive basement membrane invasion in Caenorhabditis elegans.

During invasion, cells breach basement membrane (BM) barriers with actin-rich protrusions. It remains unclear, however, whether actin polymerization applies pushing forces to help break through BM, or whether actin filaments play a passive role as scaffolding for targeting invasive machinery. Here, using the developmental event of anchor cell (AC) invasion in Caenorhabditis elegans, we observe that the AC deforms the BM and underlying tissue just before invasion, exerting forces in the tens of nanonewtons range. Deformation is driven by actin polymerization nucleated by the Arp2/3 complex and its activators, whereas formins and cross-linkers are dispensable. Delays in invasion upon actin regulator loss are not caused by defects in AC polarity, trafficking, or secretion, as appropriate markers are correctly localized in the AC even when actin is reduced and invasion is disrupted. Overall force production emerges from this study as one of the main tools that invading cells use to promote BM disruption in C. elegans.

Mammalian hemicentin 1 is assembled into tracks in the extracellular matrix of multiple tissues.

BACKGROUND: Hemicentins (HMCNs) are a family of extracellular matrix proteins first identified in Caenorhabditis elegans, with two orthologs (HMCN1 and 2) in vertebrates. In worms, HMCN is deposited at specific sites where it forms long, fine tracks that link two tissues by connecting adjacent basement membranes (BMs). By generating CRISPR/Cas9-mediated Hmcn1 and Hmcn2 knockout mice, we tested the hypothesis that HMCNs perform similar functions in mammals. RESULTS: Hmcn1 -/- mice were viable and fertile. Using new, knockout mouse-validated HMCN1 antibodies, HMCN1 was detected in wild-type mice as fine tracks along the BM of hair and whisker follicles, in the sclera of the eyes, and in the lumen of some lymphoid conduits. It was also observed in the mesangial matrix of the kidney glomerulus. However, HMCN1 deficiency did not affect the functions of these tissues, including adherence of coat hairs and whiskers, the sieving function of lymphoid conduits, or the immune response to injected antigens. HMCN2 deficiency did not lead to any discernible phenotypes on its own or when combined with HMCN1 deficiency. CONCLUSION: That Hmcn1 -/- , Hmcn2 -/- , and Hmcn1/2 double knockout mice did not display any overt phenotypes implicates compensation by other members of the fibulin family.

SPARC Promotes Cell Invasion In Vivo by Decreasing Type IV Collagen Levels in the Basement Membrane.

Overexpression of SPARC, a collagen-binding glycoprotein, is strongly associated with tumor invasion through extracellular matrix in many aggressive cancers. SPARC regulates numerous cellular processes including integrin-mediated cell adhesion, cell signaling pathways, and extracellular matrix assembly; however, the mechanism by which SPARC promotes cell invasion in vivo remains unclear. A main obstacle in understanding SPARC function has been the difficulty of visualizing and experimentally examining the dynamic interactions between invasive cells, extracellular matrix and SPARC in native tissue environments. Using the model of anchor cell invasion through the basement membrane (BM) extracellular matrix in Caenorhabditis elegans, we find that SPARC overexpression is highly pro-invasive and rescues BM transmigration in mutants with defects in diverse aspects of invasion, including cell polarity, invadopodia formation, and matrix metalloproteinase expression. By examining BM assembly, we find that overexpression of SPARC specifically decreases levels of BM type IV collagen, a crucial structural BM component. Reduction of type IV collagen mimicked SPARC overexpression and was sufficient to promote invasion. Tissue-specific overexpression and photobleaching experiments revealed that SPARC acts extracellularly to inhibit collagen incorporation into BM. By reducing endogenous SPARC, we also found that SPARC functions normally to traffic collagen from its site of synthesis to tissues that do not express collagen. We propose that a surplus of SPARC disrupts extracellular collagen trafficking and reduces BM collagen incorporation, thus weakening the BM barrier and dramatically enhancing its ability to be breached by invasive cells.

A Sensitized Screen for Genes Promoting Invadopodia Function In Vivo: CDC-42 and Rab GDI-1 Direct Distinct Aspects of Invadopodia Formation.

Invadopodia are specialized membrane protrusions composed of F-actin, actin regulators, signaling proteins, and a dynamically trafficked invadopodial membrane that drive cell invasion through basement membrane (BM) barriers in development and cancer. Due to the challenges of studying invasion in vivo, mechanisms controlling invadopodia formation in their native environments remain poorly understood. We performed a sensitized genome-wide RNAi screen and identified 13 potential regulators of invadopodia during anchor cell (AC) invasion into the vulval epithelium in C. elegans. Confirming the specificity of this screen, we identified the Rho GTPase cdc-42, which mediates invadopodia formation in many cancer cell lines. Using live-cell imaging, we show that CDC-42 localizes to the AC-BM interface and is activated by an unidentified vulval signal(s) that induces invasion. CDC-42 is required for the invasive membrane localization of WSP-1 (N-WASP), a CDC-42 effector that promotes polymerization of F-actin. Loss of CDC-42 or WSP-1 resulted in fewer invadopodia and delayed BM breaching. We also characterized a novel invadopodia regulator, gdi-1 (Rab GDP dissociation inhibitor), which mediates membrane trafficking. We show that GDI-1 functions in the AC to promote invadopodia formation. In the absence of GDI-1, the specialized invadopodial membrane was no longer trafficked normally to the invasive membrane, and instead was distributed to plasma membrane throughout the cell. Surprisingly, the pro-invasive signal(s) from the vulval cells also controls GDI-1 activity and invadopodial membrane trafficking. These studies represent the first in vivo screen for genes regulating invadopodia and demonstrate that invadopodia formation requires the integration of distinct cellular processes that are coordinated by an extracellular cue.

Nonselective autophagy reduces mitochondrial content during starvation in Caenorhabditis elegans.

Starvation significantly alters cellular physiology, and signs of aging have been reported to occur during starvation. Mitochondria are essential to the regulation of cellular energetics and aging. We sought to determine whether mitochondria exhibit signs of aging during starvation and whether quality control mechanisms regulate mitochondrial physiology during starvation. We describe effects of starvation on mitochondria in the first and third larval stages of the nematode Caenorhabditis elegans. When starved, C. elegans larvae enter developmental arrest. We observed fragmentation of the mitochondrial network, a reduction in mitochondrial DNA (mtDNA) copy number, and accumulation of DNA damage during starvation-induced developmental arrest. Mitochondrial function was also compromised by starvation. Starved worms had lower basal, maximal, and ATP-linked respiration. These observations are consistent with reduced mitochondrial quality, similar to mitochondrial phenotypes during aging. Using pharmacological and genetic approaches, we found that worms deficient for autophagy were short-lived during starvation and recovered poorly from extended starvation, indicating sensitivity to nutrient stress. Autophagy mutants unc-51/Atg1 and atg-18/Atg18 maintained greater mtDNA content than wild-type worms during starvation, suggesting that autophagy promotes mitochondrial degradation during starvation. unc-51 mutants also had a proportionally smaller reduction in oxygen consumption rate during starvation, suggesting that autophagy also contributes to reduced mitochondrial function. Surprisingly, mutations in genes involved in mitochondrial fission and fusion as well as selective mitophagy of damaged mitochondria did not affect mitochondrial content during starvation. Our results demonstrate the profound influence of starvation on mitochondrial physiology with organismal consequences, and they show that these physiological effects are influenced by autophagy.

Identification of regulators of germ stem cell enwrapment by its niche in C. elegans.

Many stem cell niches contain support cells that increase contact with stem cells by enwrapping them in cellular processes. One example is the germ stem cell niche in C. elegans, which is composed of a single niche cell termed the distal tip cell (DTC) that extends cellular processes, constructing an elaborate plexus that enwraps germ stem cells. To identify genes required for plexus formation and to explore the function of this specialized enwrapping behavior, a series of targeted and tissue-specific RNAi screens were performed. Here we identify genes that promote stem cell enwrapment by the DTC plexus, including a set that specifically functions within the DTC, such as the chromatin modifier lin-40/MTA1, and others that act within the germline, such as the 14-3-3 signaling protein par-5. Analysis of genes that function within the germline to mediate plexus development reveal that they are required for expansion of the germ progenitor zone, supporting the emerging idea that germ stem cells signal to the niche to stimulate enwrapping behavior. Examination of wild-type animals with asymmetric plexus formation and animals with reduced DTC plexus elaboration via loss of two candidates including lin-40 indicate that cellular enwrapment promotes GLP-1/Notch signaling and germ stem cell fate. Together, our work identifies novel regulators of cellular enwrapment and suggests that reciprocal signaling between the DTC niche and the germ stem cells promotes enwrapment behavior and stem cell fate.

RAB-10-Dependent Membrane Transport Is Required for Dendrite Arborization.

Formation of elaborately branched dendrites is necessary for the proper input and connectivity of many sensory neurons. Previous studies have revealed that dendritic growth relies heavily on ER-to-Golgi transport, Golgi outposts and endocytic recycling. How new membrane and associated cargo is delivered from the secretory and endosomal compartments to sites of active dendritic growth, however, remains unknown. Using a candidate-based genetic screen in C. elegans, we have identified the small GTPase RAB-10 as a key regulator of membrane trafficking during dendrite morphogenesis. Loss of rab-10 severely reduced proximal dendritic arborization in the multi-dendritic PVD neuron. RAB-10 acts cell-autonomously in the PVD neuron and localizes to the Golgi and early endosomes. Loss of function mutations of the exocyst complex components exoc-8 and sec-8, which regulate tethering, docking and fusion of transport vesicles at the plasma membrane, also caused proximal dendritic arborization defects and led to the accumulation of intracellular RAB-10 vesicles. In rab-10 and exoc-8 mutants, the trans-membrane proteins DMA-1 and HPO-30, which promote PVD dendrite stabilization and branching, no longer localized strongly to the proximal dendritic membranes and instead were sequestered within intracellular vesicles. Together these results suggest a crucial role for the Rab10 GTPase and the exocyst complex in controlling membrane transport from the secretory and/or endosomal compartments that is required for dendritic growth.

An active role for basement membrane assembly and modification in tissue sculpting.

Basement membranes are a dense, sheet-like form of extracellular matrix (ECM) that underlie epithelia and endothelia, and surround muscle, fat and Schwann cells. Basement membranes separate tissues and protect them from mechanical stress. Although traditionally thought of as a static support structure, a growing body of evidence suggests that dynamic basement membrane deposition and modification instructs coordinated cellular behaviors and acts mechanically to sculpt tissues. In this Commentary, we highlight recent studies that support the idea that far from being a passive matrix, basement membranes play formative roles in shaping tissues.

MIG-10 (lamellipodin) has netrin-independent functions and is a FOS-1A transcriptional target during anchor cell invasion in C. elegans.

To transmigrate basement membrane, cells must coordinate distinct signaling activities to breach and pass through this dense extracellular matrix barrier. Netrin expression and activity are strongly associated with invasion in developmental and pathological processes, but how netrin signaling is coordinated with other pathways during invasion is poorly understood. Using the model of anchor cell (AC) invasion in C. elegans, we have previously shown that the integrin receptor heterodimer INA-1/PAT-3 promotes netrin receptor UNC-40 (DCC) localization to the invasive cell membrane of the AC. UNC-6 (netrin)/UNC-40 interactions generate an invasive protrusion that crosses the basement membrane. To understand how UNC-40 signals during invasion, we have used genetic, site of action and live-cell imaging studies to examine the roles of known effectors of UNC-40 signaling in axon outgrowth during AC invasion. UNC-34 (Ena/VASP), the Rac GTPases MIG-2 and CED-10 and the actin binding protein UNC-115 (abLIM) are dedicated UNC-40 effectors that are recruited to the invasive membrane by UNC-40 and generate F-actin. MIG-10 (lamellipodin), an effector of UNC-40 in neurons, however, has independent functions from UNC-6/UNC-40. Furthermore, unlike other UNC-40 effectors, its expression is regulated by FOS-1A, a transcription factor that promotes basement membrane breaching. Similar to UNC-40, however, MIG-10 localization to the invasive cell membrane is also dependent on the integrin INA-1/PAT-3. These studies indicate that MIG-10 has distinct functions from UNC-40 signaling in cell invasion, and demonstrate that integrin coordinates invasion by localizing these molecules to the cell-basement membrane interface.

Repurposing an endogenous degradation system for rapid and targeted depletion of C. elegans proteins.

The capability to conditionally inactivate gene function is essential for understanding the molecular basis of development. In gene and mRNA targeting approaches, protein products can perdure, complicating genetic analysis. Current methods for selective protein degradation require drug treatment or take hours for protein removal, limiting their utility in studying rapid developmental processes in vivo. Here, we repurpose an endogenous protein degradation system to rapidly remove targeted C. elegans proteins. We show that upon expression of the E3 ubiquitin ligase substrate-recognition subunit ZIF-1, proteins tagged with the ZF1 zinc-finger domain can be quickly degraded in all somatic cell types examined with temporal and spatial control. We demonstrate that genes can be engineered to become conditional loss-of-function alleles by introducing sequences encoding the ZF1 tag into endogenous loci. Finally, we use ZF1 tagging to establish the site of cdc-42 gene function during a cell invasion event. ZF1 tagging provides a powerful new tool for the analysis of dynamic developmental events.

Identification of late larval stage developmental checkpoints in Caenorhabditis elegans regulated by insulin/IGF and steroid hormone signaling pathways.

Organisms in the wild develop with varying food availability. During periods of nutritional scarcity, development may slow or arrest until conditions improve. The ability to modulate developmental programs in response to poor nutritional conditions requires a means of sensing the changing nutritional environment and limiting tissue growth. The mechanisms by which organisms accomplish this adaptation are not well understood. We sought to study this question by examining the effects of nutrient deprivation on Caenorhabditis elegans development during the late larval stages, L3 and L4, a period of extensive tissue growth and morphogenesis. By removing animals from food at different times, we show here that specific checkpoints exist in the early L3 and early L4 stages that systemically arrest the development of diverse tissues and cellular processes. These checkpoints occur once in each larval stage after molting and prior to initiation of the subsequent molting cycle. DAF-2, the insulin/insulin-like growth factor receptor, regulates passage through the L3 and L4 checkpoints in response to nutrition. The FOXO transcription factor DAF-16, a major target of insulin-like signaling, functions cell-nonautonomously in the hypodermis (skin) to arrest developmental upon nutrient removal. The effects of DAF-16 on progression through the L3 and L4 stages are mediated by DAF-9, a cytochrome P450 ortholog involved in the production of C. elegans steroid hormones. Our results identify a novel mode of C. elegans growth in which development progresses from one checkpoint to the next. At each checkpoint, nutritional conditions determine whether animals remain arrested or continue development to the next checkpoint.

Basement Membranes in the Worm: A Dynamic Scaffolding that Instructs Cellular Behaviors and Shapes Tissues.

The nematode worm Caenorhabditis elegans has all the major basement membrane proteins found in vertebrates, usually with a smaller gene family encoding each component. With its powerful forward genetics, optical clarity, simple tissue organization, and the capability to functionally tag most basement membrane components with fluorescent proteins, C. elegans has facilitated novel insights into the assembly and function of basement membranes. Although basement membranes are generally thought of as static structures, studies in C. elegans have revealed their active properties and essential functions in tissue formation and maintenance. Here, we review discoveries from C. elegans development that highlight dynamic aspects of basement membrane assembly, function, and regulation during organ growth, tissue polarity, cell migration, cell invasion, and tissue attachment. These studies have helped transform our view of basement membranes from static support structures to dynamic scaffoldings that play broad roles in regulating tissue organization and cellular behavior that are essential for development and have important implications in human diseases.

Basement membrane dynamics in living animals: Insights and pitfalls.

Recent studies with fluorophore-tagged basement membrane (BM) components have led to remarkable discoveries about BMs but also inconsistent interpretations. Here, we review types of BM dynamics, discuss how we conduct and interpret fluorophore-tagged BM studies, and highlight experimental conditions that are important to consider.

Post-embryonic endogenous expression and localization of LET-60/Ras in C. elegans.

Ras GTPases regulate many developmental and physiological processes and mutations in Ras are associated with numerous human cancers. Here, we report the function, levels, and localization of an N-terminal knock-in of mNeonGreen (mNG) into C. elegans LET-60 /Ras. mNG:: LET-60 interferes with some but not all LET-60 /Ras functions. mNG:: LET-60 is broadly present in tissues, found at different levels in cells, and concentrates in distinct subcellular compartments, including the nucleolus, nucleus, intracellular region, and plasma membrane. These results suggest that mNG:: LET-60 can be a useful tool for determining LET-60 levels and localization once its functionality in a developmental or physiological process is established.

Translational regulation of cell invasion through extracellular matrix-an emerging role for ribosomes.

Many developmental and physiological processes require cells to invade and migrate through extracellular matrix barriers. This specialized cellular behavior is also misregulated in many diseases, such as immune disorders and cancer. Cell invasive activity is driven by pro-invasive transcriptional networks that activate the expression of genes encoding numerous different proteins that expand and regulate the cytoskeleton, endomembrane system, cell adhesion, signaling pathways, and metabolic networks. While detailed mechanistic studies have uncovered crucial insights into pro-invasive transcriptional networks and the distinct cell biological attributes of invasive cells, less is known about how invasive cells modulate mRNA translation to meet the robust, dynamic, and unique protein production needs of cell invasion. In this review we outline known modes of translation regulation promoting cell invasion and focus on recent studies revealing elegant mechanisms that expand ribosome biogenesis within invasive cells to meet the increased protein production requirements to invade and migrate through extracellular matrix barriers.

Chronic high-sugar diet in adulthood protects Caenorhabditis elegans from 6-OHDA induced dopaminergic neurodegeneration.

BACKGROUND: Diets high in saturated fat and sugar, termed western diets, have been associated with several negative health outcomes, including increased risk for neurodegenerative disease. Parkinson s Disease (PD) is the second most prevalent neurodegenerative disease and is characterized by the progressive death of dopaminergic neurons in the brain. We build upon previous work characterizing the impact of high sugar diets in Caenorhabditis elegans to mechanistically evaluate the relationship between high sugar diets and dopaminergic neurodegeneration. RESULTS: Non-developmental high glucose and fructose diets led to increased lipid content and shorter lifespan and decreased reproduction. However, in contrast to previous reports, we found that non-developmental chronic high-glucose and high-fructose diets did not induce dopaminergic neurodegeneration alone and were protective from 6-hydroxydopamine (6-OHDA) induced degeneration. Neither sugar altered baseline electron transport chain function, and both increased vulnerability to organism-wide ATP depletion when the electron transport chain was inhibited, arguing against energetic rescue as a basis for neuroprotection. The induction of oxidative stress by 6-OHDA is hypothesized to contribute to its pathology, and high sugar diets prevented this increase in the soma of the dopaminergic neurons. However, we did not find increased expression of antioxidant enzymes or glutathione levels. Instead, we found evidence suggesting alterations to dopamine transmission that could result in decreased 6-OHDA uptake. CONCLUSION: Our work uncovers a neuroprotective role for high sugar diets, despite concomitant decreases in lifespan and reproduction. Our results support the broader finding that ATP depletion alone is insufficient to induce dopaminergic neurodegeneration, whereas increased neuronal oxidative stress may drive degeneration. Finally, our work highlights the importance of evaluating lifestyle by toxicant interactions.

Hemicentin-mediated type IV collagen assembly strengthens juxtaposed basement membrane linkage.

Basement membrane (BM) matrices surround and separate most tissues. However, through poorly understood mechanisms, BMs of adjacent tissue can also stably link to support organ structure and function. Using endogenous knock-in fluorescent proteins, conditional RNAi, optogenetics, and quantitative live imaging, we identified extracellular matrix proteins mediating a BM linkage (B-LINK) between the uterine utse and epidermal seam cell BMs in Caenorhabditis elegans that supports the uterus during egg-laying. We found that hemicentin is secreted by the utse and promotes fibulin-1 assembly to jointly initiate the B-LINK. During egg-laying, however, both proteins' levels decline and are not required for B-LINK maintenance. Instead, we discovered that hemicentin recruits ADAMTS9/20, which facilitates the assembly of high levels of type IV collagen that sustains the B-LINK during the mechanically active egg-laying period. This work reveals mechanisms underlying BM-BM linkage maturation and identifies a crucial function for hemicentin and fibulin-1 in initiating attachment and type IV collagen in strengthening this specialized form of tissue linkage.

Reciprocal discoidin domain receptor signaling strengthens integrin adhesion to connect adjacent tissues.

Separate tissues connect through adjoining basement membranes to carry out molecular barrier, exchange, and organ support functions. Cell adhesion at these connections must be robust and balanced to withstand independent tissue movement. Yet, how cells achieve synchronized adhesion to connect tissues is unknown. Here, we have investigated this question using the Caenorhabditis elegans utse-seam tissue connection that supports the uterus during egg-laying. Through genetics, quantitative fluorescence, and cell-specific molecular disruption, we show that type IV collagen, which fastens the linkage, also activates the collagen receptor discoidin domain receptor-2 (DDR-2) in both the utse and seam. RNAi depletion, genome editing, and photobleaching experiments revealed that DDR-2 signals through LET-60/Ras to coordinately strengthen an integrin adhesion in the utse and seam that stabilizes their connection. These results uncover a synchronizing mechanism for robust adhesion during tissue connection, where collagen both affixes the linkage and signals to both tissues to bolster their adhesion.