The Experts below are selected from a list of 18912 Experts worldwide ranked by ideXlab platform
David R. Sherwood - One of the best experts on this subject based on the ideXlab platform.
-
live Cell confocal microscopy and quantitative 4d image analysis of Anchor Cell invasion through the basement membrane in caenorhabditis elegans
Nature Protocols, 2017Co-Authors: Laura C Kelley, Elliott J. Hagedorn, Zheng Wang, Sam A. Johnson, Lin Wang, Wanqing Shen, Shijun Lei, David R. SherwoodAbstract:Cell invasion through basement membrane (BM) barriers is crucial in development, leukocyte trafficking and the spread of cancer. The mechanisms that direct invasion, despite their importance in normal and disease states, are poorly understood, largely because of the inability to visualize dynamic Cell-BM interactions in vivo. This protocol describes multichannel time-lapse confocal imaging of Anchor-Cell invasion in live Caenorhabditis elegans. Methods presented include outline-slide preparation and worm growth synchronization (15 min), mounting (20 min), image acquisition (20-180 min), image processing (20 min) and quantitative analysis (variable timing). The acquired images enable direct measurement of invasive dynamics including formation of invadopodia and Cell-membrane protrusions, and removal of BM. This protocol can be combined with genetic analysis, molecular-activity probes and optogenetic approaches to uncover the molecular mechanisms underlying Cell invasion. These methods can also be readily adapted by any worm laboratory for real-time analysis of Cell migration, BM turnover and Cell-membrane dynamics.
-
breaching and opening basement membrane barriers the Anchor Cell leads the way
2017Co-Authors: Daniel P Keeley, David R. SherwoodAbstract:Metastasis is initiated in epithelial-derived tumors when Cells at the tumor front breach the epithelial basement membrane (BM). Invasion through BMs is thought to be one of the most rate-limiting steps in cancer progression and thus is a therapeutically attractive target for halting tumor spread. Despite intense interest, it has been challenging to experimentally determine how invasive Cells breach and clear BM barriers, which has hindered efforts to block metastasis. Here we discuss how an experimentally tractable developmental invasion event, Anchor Cell (AC) invasion in the model system C. elegans, is offering powerful new insights into the fundamental mechanisms that invasive Cells use to breach BM barriers and how Cells at the breach site widen BM gaps through a new mechanism called BM sliding. Finally, we cover studies demonstrating that AC invasion can also be used as a new paradigm to examine how alterations in the tumor microenvironment impinge on Cell invasive behavior.
-
a sensitized screen for genes promoting invadopodia function in vivo cdc 42 and rab gdi 1 direct distinct aspects of invadopodia formation
PLOS Genetics, 2016Co-Authors: Lauren L Lohmer, Elliott J. Hagedorn, Joshua W. Ziel, Qiuyi Chi, Matthew R Clay, Kaleb M Naegeli, Jieun Park, Ranjay Jayadev, David R. SherwoodAbstract: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.
-
unc 6 netrin stabilizes oscillatory clustering of the unc 40 dcc receptor to orient polarity
Journal of Cell Biology, 2014Co-Authors: Zheng Wang, Elliott J. Hagedorn, Joshua W. Ziel, Lara M. Linden, Qiuyi Chi, Kaleb M Naegeli, Natasha S Savage, David R. SherwoodAbstract:The receptor deleted in colorectal cancer (DCC) directs dynamic polarizing activities in animals toward its extraCellular ligand netrin. How DCC polarizes toward netrin is poorly understood. By performing live-Cell imaging of the DCC orthologue UNC-40 during Anchor Cell invasion in Caenorhabditis elegans, we have found that UNC-40 clusters, recruits F-actin effectors, and generates F-actin in the absence of UNC-6 (netrin). Time-lapse analyses revealed that UNC-40 clusters assemble, disassemble, and reform at periodic intervals in different regions of the Cell membrane. This oscillatory behavior indicates that UNC-40 clusters through a mechanism involving interlinked positive (formation) and negative (disassembly) feedback. We show that endogenous UNC-6 and ectopically provided UNC-6 orient and stabilize UNC-40 clustering. Furthermore, the UNC-40–binding protein MADD-2 (a TRIM family protein) promotes ligand-independent clustering and robust UNC-40 polarization toward UNC-6. Together, our data suggest that UNC-6 (netrin) directs polarized responses by stabilizing UNC-40 clustering. We propose that ligand-independent UNC-40 clustering provides a robust and adaptable mechanism to polarize toward netrin.
-
mig 10 lamellipodin has netrin independent functions and is a fos 1a transcriptional target during Anchor Cell invasion in c elegans
Development, 2014Co-Authors: Zheng Wang, Qiuyi Chi, David R. SherwoodAbstract: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.
Paul W. Sternberg - One of the best experts on this subject based on the ideXlab platform.
-
Anchor Cell signaling and vulval precursor Cell positioning establish a reproducible spatial context during c elegans vulval induction
Developmental Biology, 2016Co-Authors: Stephanie Grimbert, Paul W. Sternberg, Kyria Tietze, Michalis Barkoulas, Marieanne Felix, Christian BraendleAbstract:How Cells coordinate their spatial positioning through interCellular signaling events is poorly understood. Here we address this topic using Caenorhabditis elegans vulval patterning during which hypodermal vulval precursor Cells (VPCs) adopt distinct Cell fates determined by their relative positions to the gonadal Anchor Cell (AC). LIN-3/EGF signaling by the AC induces the central VPC, P6.p, to adopt a 1° vulval fate. Exact alignment of AC and VPCs is thus critical for correct fate patterning, yet, as we show here, the initial AC-VPC positioning is both highly variable and asymmetric among individuals, with AC and P6.p only becoming aligned at the early L3 stage. Cell ablations and mutant analysis indicate that VPCs, most prominently 1° Cells, move towards the AC. We identify AC-released LIN-3/EGF as a major attractive signal, which therefore plays a dual role in vulval patterning (Cell alignment and fate induction). Additionally, compromising Wnt pathway components also induces AC-VPC alignment errors, with loss of posterior Wnt signaling increasing stochastic vulval centering on P5.p. Our results illustrate how interCellular signaling reduces initial spatial variability in Cell positioning to generate reproducible interactions across tissues.
-
fos 1 promotes basement membrane removal during Anchor Cell invasion in c elegans
Cell, 2005Co-Authors: David R. Sherwood, James Butler, James M Kramer, Paul W. SternbergAbstract:Cell invasion through basement membranes is crucial during morphogenesis and cancer metastasis. Here, we genetically dissect this process during Anchor-Cell invasion into the vulval epithelium in C. elegans. We have identified the fos transcription factor ortholog fos-1 as a critical regulator of basement-membrane removal. In fos-1 mutants, the gonadal Anchor Cell extends Cellular processes normally toward vulval Cells, but these processes fail to remove the basement membranes separating the gonad from the vulval epithelium. fos-1 is expressed in the Anchor Cell and controls invasion Cell autonomously. We have identified ZMP-1, a membrane-type matrix metalloproteinase, CDH-3, a Fat-like protocadherin, and hemicentin, a fibulin family extraCellular matrix protein, as transcriptional targets of FOS-1 that promote invasion. These results reveal a key genetic network that controls basement-membrane removal during Cell invasion.
-
a Cell specific enhancer that specifies lin 3 expression in the c elegans Anchor Cell for vulval development
Development, 2004Co-Authors: Byung Joon Hwang, Paul W. SternbergAbstract:During C. elegans vulval development, the Anchor Cell (AC) in the somatic gonad expresses lin-3, activating the EGF receptor signaling pathway in vulval precursor Cells (VPCs) and thereby inducing and patterning VPCs. Previous studies with lin-3 mutants and transgene expression have revealed that the level of LIN-3 in the AC must be precisely regulated for proper vulval development. To understand how lin-3 expression is achieved in the AC, we identified a 59 bp lin-3 enhancer sufficient to activate lin-3 transcription solely in the AC. The enhancer contains two E-box elements, and one FTZ-F1 nuclear hormone receptor (NHR) binding site that is mutated in a vulvaless mutant, lin-3(e1417). Mutagenesis studies show that both E-boxes and the NHR binding site are necessary to express lin-3 in the AC. In vitro DNA-binding studies and in vivo functional assays indicate that distinct trans-acting factors, including the E-protein/Daughterless homolog HLH-2 and unidentified nuclear hormone receptor(s), are necessary for lin-3 transcription in the AC and thus are involved in vulval development.
-
Anchor Cell invasion into the vulval epithelium in c elegans
Developmental Cell, 2003Co-Authors: David R. Sherwood, Paul W. SternbergAbstract:An understanding of Cell-invasive behavior has been limited by the lack of in vivo models where this activity can be clearly visualized and manipulated. We show that a single Cell in the Caenorhabditis elegans gonad, the Anchor Cell (AC), initiates uterine-vulval contact through a Cell invasion event. Using genetic analysis, laser ablations, and Cell-specific markers, we demonstrate that AC invasion is predominantly stimulated by the 1 degrees vulval lineage Cells, which generate a diffusible signal that promotes AC invasive behavior toward these Cells and further targets invasive processes between the two central 1 degrees vulval lineage Cells. We also show that AC invasion is regulated by the AC response to this cue, as well as a vulval-independent mechanism that weakly drives invasion. These studies dissect the regulatory mechanisms that underlie a simple Cell-invasive behavior in vivo, and introduce AC invasion as a model for understanding key checkpoints controlling Cell invasion.
-
cis regulatory control of three Cell fate specific genes in vulval organogenesis of caenorhabditis elegans and c briggsae
Developmental Biology, 2003Co-Authors: Martha Kirouac, Paul W. SternbergAbstract:The great-grandprogeny of the Caenorhabditis elegans vulval precursor Cells (VPCs) adopt one of the final vulA, B1, B2, C, D, E, and F Cell fates in a precise spatial pattern. This pattern of vulval Cell types is likely to depend on the cis-regulatory regions of the transcriptional targets of interCellular signals in vulval development. egl-17, zmp-1, and cdh-3 are expressed differentially in the developing vulva Cells, providing a potential readout for different signaling pathways. To understand how such pathways interact to specify unique vulval Cell types in a precise pattern, we have identified cis-regulatory regions sufficient to confer vulval Cell type-specific regulation when fused in cis to the basal pes-10 promoter. We have identified the C. briggsae homologs of these three genes, with their corresponding control regions, and tested these regions in both C. elegans and C. briggsae. These regions of similarity in C. elegans and C. briggsae upstream of egl-17, zmp-1, and cdh-3 promote expression in vulval Cells and the Anchor Cell (AC). By using the cis-regulatory analysis and phylogenetic footprinting, we have identified overrepresented sequences involved in conferring vulval and AC expression.
Elliott J. Hagedorn - One of the best experts on this subject based on the ideXlab platform.
-
live Cell confocal microscopy and quantitative 4d image analysis of Anchor Cell invasion through the basement membrane in caenorhabditis elegans
Nature Protocols, 2017Co-Authors: Laura C Kelley, Elliott J. Hagedorn, Zheng Wang, Sam A. Johnson, Lin Wang, Wanqing Shen, Shijun Lei, David R. SherwoodAbstract:Cell invasion through basement membrane (BM) barriers is crucial in development, leukocyte trafficking and the spread of cancer. The mechanisms that direct invasion, despite their importance in normal and disease states, are poorly understood, largely because of the inability to visualize dynamic Cell-BM interactions in vivo. This protocol describes multichannel time-lapse confocal imaging of Anchor-Cell invasion in live Caenorhabditis elegans. Methods presented include outline-slide preparation and worm growth synchronization (15 min), mounting (20 min), image acquisition (20-180 min), image processing (20 min) and quantitative analysis (variable timing). The acquired images enable direct measurement of invasive dynamics including formation of invadopodia and Cell-membrane protrusions, and removal of BM. This protocol can be combined with genetic analysis, molecular-activity probes and optogenetic approaches to uncover the molecular mechanisms underlying Cell invasion. These methods can also be readily adapted by any worm laboratory for real-time analysis of Cell migration, BM turnover and Cell-membrane dynamics.
-
a sensitized screen for genes promoting invadopodia function in vivo cdc 42 and rab gdi 1 direct distinct aspects of invadopodia formation
PLOS Genetics, 2016Co-Authors: Lauren L Lohmer, Elliott J. Hagedorn, Joshua W. Ziel, Qiuyi Chi, Matthew R Clay, Kaleb M Naegeli, Jieun Park, Ranjay Jayadev, David R. SherwoodAbstract: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.
-
unc 6 netrin stabilizes oscillatory clustering of the unc 40 dcc receptor to orient polarity
Journal of Cell Biology, 2014Co-Authors: Zheng Wang, Elliott J. Hagedorn, Joshua W. Ziel, Lara M. Linden, Qiuyi Chi, Kaleb M Naegeli, Natasha S Savage, David R. SherwoodAbstract:The receptor deleted in colorectal cancer (DCC) directs dynamic polarizing activities in animals toward its extraCellular ligand netrin. How DCC polarizes toward netrin is poorly understood. By performing live-Cell imaging of the DCC orthologue UNC-40 during Anchor Cell invasion in Caenorhabditis elegans, we have found that UNC-40 clusters, recruits F-actin effectors, and generates F-actin in the absence of UNC-6 (netrin). Time-lapse analyses revealed that UNC-40 clusters assemble, disassemble, and reform at periodic intervals in different regions of the Cell membrane. This oscillatory behavior indicates that UNC-40 clusters through a mechanism involving interlinked positive (formation) and negative (disassembly) feedback. We show that endogenous UNC-6 and ectopically provided UNC-6 orient and stabilize UNC-40 clustering. Furthermore, the UNC-40–binding protein MADD-2 (a TRIM family protein) promotes ligand-independent clustering and robust UNC-40 polarization toward UNC-6. Together, our data suggest that UNC-6 (netrin) directs polarized responses by stabilizing UNC-40 clustering. We propose that ligand-independent UNC-40 clustering provides a robust and adaptable mechanism to polarize toward netrin.
-
The netrin receptor DCC focuses invadopodia-driven basement membrane transmigration in vivo.
Journal of Cell Biology, 2013Co-Authors: Elliott J. Hagedorn, Meghan A. Morrissey, Joshua W. Ziel, Lara M. Linden, Zheng Wang, Qiuyi Chi, Sam A. Johnson, David R. SherwoodAbstract:Though critical to normal development and cancer metastasis, how Cells traverse basement membranes is poorly understood. A central impediment has been the challenge of visualizing invasive Cell interactions with basement membrane in vivo. By developing live-Cell imaging methods to follow Anchor Cell (AC) invasion in Caenorhabditis elegans, we identify F-actin–based invadopodia that breach basement membrane. When an invadopodium penetrates basement membrane, it rapidly transitions into a stable invasive process that expands the breach and crosses into the vulval tissue. We find that the netrin receptor UNC-40 (DCC) specifically enriches at the site of basement membrane breach and that activation by UNC-6 (netrin) directs focused F-actin formation, generating the invasive protrusion and the cessation of invadopodia. Using optical highlighting of basement membrane components, we further demonstrate that rather than relying solely on proteolytic dissolution, the AC’s protrusion physically displaces basement membrane. These studies reveal an UNC-40–mediated morphogenetic transition at the Cell–basement membrane interface that directs invading Cells across basement membrane barriers.
-
Cell invasion through basement membrane: The netrin receptor DCC guides the way.
Worm, 2013Co-Authors: Meghan A. Morrissey, Elliott J. Hagedorn, David R. SherwoodAbstract:Cell invasion through basement membrane is an essential part of normal development and physiology, and occurs during the pathological progression of human inflammatory diseases and cancer. F-actin-rich membrane protrusions, called invadopodia, have been hypothesized to be the “drill bits” of invasive Cells, mediating invasion through the dense, highly cross-linked basement membrane matrix. Though studied in vitro for over 30 y, invadopodia function in vivo has remained elusive. We have recently discovered that invadopodia breach basement membrane during Anchor Cell invasion in C. elegans, a genetically and visually tractable in vivo invasion event. Further, we found that the netrin receptor DCC localizes to the initial site of basement membrane breach and directs invasion through a single gap in the matrix. In this commentary, we examine how the dynamics and structure of AC-invadopodia compare with in vitro invadopodia and how the netrin receptor guides invasion through a single basement membrane breach. We end with a discussion of our surprising result that the Anchor Cell pushes the basement membrane aside, instead of completely dissolving it through proteolysis, and provide some ideas for how proteases and physical displacement may work together to ensure efficient and robust invasion.
Alex Hajnal - One of the best experts on this subject based on the ideXlab platform.
-
reciprocal egfr signaling in the Anchor Cell ensures precise inter organ connection during c elegans vulval morphogenesis
bioRxiv, 2021Co-Authors: Silvan Spiri, Louisa Mereu, Simon Berger, Andrew J Demello, Alex HajnalAbstract:During C. elegans vulval development, the uterine Anchor Cell (AC) first secretes an epidermal growth factor (EGF) to specify the vulval Cell fates and then invades into the underlying vulval epithelium. Thereby, the AC establishes direct contact with the invaginating 1{degrees} vulF Cells and attaches the developing uterus to the vulva. The signals involved and the exact sequence of events joining these two organs are not fully understood. Using a conditional let-23 egf receptor (EGFR) allele along with novel microfluidic short- and long-term imaging methods, we discovered a specific function of the EGFR in the AC during vulval lumen morphogenesis. Tissue-specific inactivation of let-23 in the AC resulted in imprecise alignment of the AC with the 1{degrees} vulval Cells, delayed AC invasion and disorganized adherens junctions at the newly forming contact site between the AC and the dorsal vulF toroid. We propose that EGFR signaling, activated by a reciprocal EGF cue from the 1{degrees} vulval Cells, positions the AC at the vulval midline, guides it during invasion and assembles a cytoskeletal scaffold organizing the adherens junctions that connect the developing uterus to the dorsal vulF toroid. EGFR signaling in the AC thus ensures the precise alignment of the two developing organs.
-
the invading Anchor Cell induces lateral membrane constriction during vulval lumen morphogenesis in c elegans
Developmental Cell, 2017Co-Authors: Daniel Roiz, Qiutan Yang, Louisa Mereu, Michael Daube, Alex HajnalAbstract:Summary During epithelial tube morphogenesis, linear arrays of Cells are converted into tubular structures through actomyosin-generated intraCellular forces that induce tissue invagination and lumen formation. We have investigated lumen morphogenesis in the C. elegans vulva. The first discernible event initiating lumen formation is the apical constriction of the two innermost primary Cells (VulF). The VulF Cells thereafter constrict their lateral membranes along the apicobasal axis to extend the lumen dorsally. Lateral, but not apical, VulF constriction requires the prior invasion of the Anchor Cell (AC). The invading AC extends actin-rich protrusions toward VulF, resulting in the formation of a direct AC-VulF interface. The recruitment of the F-BAR-domain protein TOCA-1 to the AC-VulF interface induces the accumulation of force-generating actomyosin, causing a switch from apical to lateral membrane constriction and the dorsal extension of the lumen. Invasive Cells may induce shape changes in adjacent Cells to penetrate their target tissues.
-
the caenorhabditis elegans homolog of the opitz syndrome gene madd 2 mid1 regulates Anchor Cell invasion during vulval development
Developmental Biology, 2013Co-Authors: Matthias K Morf, Ivo Rimann, Mariam Alexander, Peter J Roy, Alex HajnalAbstract:Mutations in the human Mid1 gene cause Opitz G/BBB syndrome, which is characterized by various midline closure defects. The Caenorhabditis elegans homolog of Mid1, madd-2, positively regulates signaling by the unc-40 Netrin receptor during the extension of muscle arms to the midline and in axon guidance and branching. During uterine development, a specialized Cell called Anchor Cell (AC) breaches the basal laminae separating the uterus from the epidermis and invades the underlying vulval tissue. AC invasion is guided by an UNC-6 Netrin signal from the ventral nerve cord and an unknown guidance signal from the vulval Cells. Using genetic epistasis analysis, we show that madd-2 regulates AC invasion downstream of or in parallel with the Netrin signaling pathway. Measurements of AC shape, polarity and dynamics indicate that MADD-2 prevents the formation of ectopic AC protrusions in the absence of guidance signals. We propose that MADD-2 represses the intrinsic invasive capacity of the AC, while the Netrin and vulval guidance cues locally overcome this inhibitory activity of MADD-2 to guide the AC ventrally into the vulval tissue. Therefore, developmental Cell invasion depends on a precise balance between pro- and anti-invasive factors.
-
regulation of Anchor Cell invasion and uterine Cell fates by the egl 43 evi 1 proto oncogene in caenorhabditis elegans
Developmental Biology, 2007Co-Authors: Ivo Rimann, Alex HajnalAbstract:Cell invasion is a tightly controlled process occurring during development and tumor progression. The nematode Caenorhabditis elegans serves as a genetic model to study Cell invasion during normal development. In the third larval stage, the Anchor Cell in the somatic gonad first induces and then invades the adjacent epidermal vulval precursor Cells. The homolog of the Evi-1 oncogene, egl-43, is necessary for basement membrane destruction and Anchor Cell invasion. egl-43 is part of a regulatory network mediating Cell invasion downstream of the fos-1 proto-oncogene. In addition, EGL-43 is required to specify the Cell fates of ventral uterus Cells downstream of or in parallel with LIN-12 NOTCH. Comparison with mammalian Evi-1 suggests a conserved pathway controlling Cell invasion and Cell fate specification.
Zheng Wang - One of the best experts on this subject based on the ideXlab platform.
-
live Cell confocal microscopy and quantitative 4d image analysis of Anchor Cell invasion through the basement membrane in caenorhabditis elegans
Nature Protocols, 2017Co-Authors: Laura C Kelley, Elliott J. Hagedorn, Zheng Wang, Sam A. Johnson, Lin Wang, Wanqing Shen, Shijun Lei, David R. SherwoodAbstract:Cell invasion through basement membrane (BM) barriers is crucial in development, leukocyte trafficking and the spread of cancer. The mechanisms that direct invasion, despite their importance in normal and disease states, are poorly understood, largely because of the inability to visualize dynamic Cell-BM interactions in vivo. This protocol describes multichannel time-lapse confocal imaging of Anchor-Cell invasion in live Caenorhabditis elegans. Methods presented include outline-slide preparation and worm growth synchronization (15 min), mounting (20 min), image acquisition (20-180 min), image processing (20 min) and quantitative analysis (variable timing). The acquired images enable direct measurement of invasive dynamics including formation of invadopodia and Cell-membrane protrusions, and removal of BM. This protocol can be combined with genetic analysis, molecular-activity probes and optogenetic approaches to uncover the molecular mechanisms underlying Cell invasion. These methods can also be readily adapted by any worm laboratory for real-time analysis of Cell migration, BM turnover and Cell-membrane dynamics.
-
unc 6 netrin stabilizes oscillatory clustering of the unc 40 dcc receptor to orient polarity
Journal of Cell Biology, 2014Co-Authors: Zheng Wang, Elliott J. Hagedorn, Joshua W. Ziel, Lara M. Linden, Qiuyi Chi, Kaleb M Naegeli, Natasha S Savage, David R. SherwoodAbstract:The receptor deleted in colorectal cancer (DCC) directs dynamic polarizing activities in animals toward its extraCellular ligand netrin. How DCC polarizes toward netrin is poorly understood. By performing live-Cell imaging of the DCC orthologue UNC-40 during Anchor Cell invasion in Caenorhabditis elegans, we have found that UNC-40 clusters, recruits F-actin effectors, and generates F-actin in the absence of UNC-6 (netrin). Time-lapse analyses revealed that UNC-40 clusters assemble, disassemble, and reform at periodic intervals in different regions of the Cell membrane. This oscillatory behavior indicates that UNC-40 clusters through a mechanism involving interlinked positive (formation) and negative (disassembly) feedback. We show that endogenous UNC-6 and ectopically provided UNC-6 orient and stabilize UNC-40 clustering. Furthermore, the UNC-40–binding protein MADD-2 (a TRIM family protein) promotes ligand-independent clustering and robust UNC-40 polarization toward UNC-6. Together, our data suggest that UNC-6 (netrin) directs polarized responses by stabilizing UNC-40 clustering. We propose that ligand-independent UNC-40 clustering provides a robust and adaptable mechanism to polarize toward netrin.
-
mig 10 lamellipodin has netrin independent functions and is a fos 1a transcriptional target during Anchor Cell invasion in c elegans
Development, 2014Co-Authors: Zheng Wang, Qiuyi Chi, David R. SherwoodAbstract: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.
-
The netrin receptor DCC focuses invadopodia-driven basement membrane transmigration in vivo.
Journal of Cell Biology, 2013Co-Authors: Elliott J. Hagedorn, Meghan A. Morrissey, Joshua W. Ziel, Lara M. Linden, Zheng Wang, Qiuyi Chi, Sam A. Johnson, David R. SherwoodAbstract:Though critical to normal development and cancer metastasis, how Cells traverse basement membranes is poorly understood. A central impediment has been the challenge of visualizing invasive Cell interactions with basement membrane in vivo. By developing live-Cell imaging methods to follow Anchor Cell (AC) invasion in Caenorhabditis elegans, we identify F-actin–based invadopodia that breach basement membrane. When an invadopodium penetrates basement membrane, it rapidly transitions into a stable invasive process that expands the breach and crosses into the vulval tissue. We find that the netrin receptor UNC-40 (DCC) specifically enriches at the site of basement membrane breach and that activation by UNC-6 (netrin) directs focused F-actin formation, generating the invasive protrusion and the cessation of invadopodia. Using optical highlighting of basement membrane components, we further demonstrate that rather than relying solely on proteolytic dissolution, the AC’s protrusion physically displaces basement membrane. These studies reveal an UNC-40–mediated morphogenetic transition at the Cell–basement membrane interface that directs invading Cells across basement membrane barriers.
-
integrin acts upstream of netrin signaling to regulate formation of the Anchor Cell s invasive membrane in c elegans
Developmental Cell, 2009Co-Authors: Elliott J. Hagedorn, Joshua W. Ziel, Zheng Wang, Hanako Yashiro, Shinji Ihara, David R. SherwoodAbstract:Integrin expression and activity have been strongly correlated with developmental and pathological processes involving Cell invasion through basement membranes. The role of integrins in mediating these invasions, however, remains unclear. Utilizing the genetically and visually accessible model of Anchor Cell (AC) invasion in C. elegans, we have recently shown that netrin signaling orients a specialized invasive Cell membrane domain toward the basement membrane. Here, we demonstrate that the integrin heterodimer INA-1/PAT-3 plays a crucial role in AC invasion, in part by targeting the netrin receptor UNC-40 (DCC) to the AC's plasma membrane. Analyses of the invasive membrane components phosphatidylinositol 4,5-bisphosphate, the Rac GTPase MIG-2, and F-actin further indicate that INA-1/PAT-3 plays a broad role in promoting the plasma membrane association of these molecules. Taken together, these studies reveal a role for integrin in regulating the plasma membrane targeting and netrin-dependent orientation of a specialized invasive membrane domain.