TLN-1/talin is a conserved focal adhesion protein that forms part of the linkage between the cyto... more TLN-1/talin is a conserved focal adhesion protein that forms part of the linkage between the cytoplasmic tail of integrin and the actin cytoskeleton. In C. elegans , TLN-1 is expressed strongly in striated muscle and the gonadal sheath cells. Here, we report that a CRISPR-generated TLN-1 allele TLN-1(W387A), predicted to affect binding of talin to integrins, results in mild phenotypes, including motility defects and ovulation defects. The arrangement of the actin cytoskeleton in the body wall muscles, spermatheca, and sheath appears identical in wild type and TLN-1(W387A) animals. This analysis suggests that W387 in TLN-1 does not have a major effect on the binding of talin to integrin in vivo .
<p>Panel A: CKI-1::GFP expression levels were assessed in the transgenic rescued lines. Top... more <p>Panel A: CKI-1::GFP expression levels were assessed in the transgenic rescued lines. Top bands in lanes 1 and 2 show the relative level of UNC-54/myosin B in each sample. Bottom bands indicate the level of CKI-1::GFP. Lanes 1 and 2 represent <i>pat-3</i>(+) and <i>pat-3(sp)</i>, respectively. Quantification (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042425#pone.0042425.s003" target="_blank">Table S1</a>) using ImageJ software revealed that CKI-1::GFP level was 10-fold increased in <i>pat-3(sp)</i> animals compared to <i>pat-3</i>(+) animals. Panel B: CKI-1::GFP expression levels were assessed in <i>pat-3</i>(+) animals treated with RNAi directed against focal adhesion genes. Top bands represent the amount of CKI-1::GFP in extracts prepared from each RNAi condition. L4440 is a negative RNAi control. The <i>pat-3</i>, <i>ina-1</i>, <i>unc-97</i>, <i>unc-52, pat-6</i> and <i>let-2</i> RNAi caused upregulation of CKI-1::GFP, while <i>unc-112</i> RNAi had no effect. Bottom bands indicate MH33 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042425#pone.0042425-Francis1" target="_blank">[90]</a> levels in each lane as a loading control. Quantification (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042425#pone.0042425.s003" target="_blank">Table S1</a>) using ImageJ software revealed that CKI-1::GFP level was increased by RNAi of <i>pat-3</i>, <i>ina-1</i>, <i>unc-97</i>, <i>unc-52, pat-6,</i> and <i>let-2</i>. Panel C: CKI-1::GFP expression levels were also measured in <i>pat-3</i>(+) animals treated with E3 ligase gene RNAi. Top bands represent the amount of CKI-1::GFP in the extracts prepared from each RNAi condition. L4440 is a negative RNAi control. The <i>skpt-1</i>, <i>lin-23</i> and <i>cul-1</i> RNAi depletions caused upregulation of CKI-1::GFP, while <i>rbx-1</i> and <i>cul-4</i> RNAi had no effect. Bottom bands indicate MH33 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042425#pone.0042425-Francis1" target="_blank">[90]</a> levels in each lane as a loading control. Quantification (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042425#pone.0042425.s003" target="_blank">Table S1</a>) using ImageJ software revealed that the CKI-1::GFP level was increased by RNAi of <i>skpt-1</i>, <i>lin-23</i> and <i>cul-1</i>.</p
The cell-extracellular matrix (ECM) interaction plays an essential role in maintaining tissue sha... more The cell-extracellular matrix (ECM) interaction plays an essential role in maintaining tissue shapes and regulates cell behaviors such as cell adhesion, differentiation and proliferation. The mechanism by which the ECM influences the cell cycle in vivo is poorly understood. Here we demonstrate that the b integrin PAT-3 regulates the localization and expression of CKI-1, a C. elegans homologue of the cyclin dependent kinase inhibitor p27KIP1. In nematodes expressing wild type PAT-3, CKI-1::GFP localizes primarily to nucleoli in hypodermal cells, whereas in animals expressing mutant pat-3 with a defective splice junction, CKI-1::GFP appears clumped and disorganized in nucleoplasm. RNAi analysis links cell adhesion genes to the regulation of CKI-1. RNAi of unc-52/perlecan, ina-1/a integrin, pat-4/ILK, and unc-97/PINCH resulted in abnormal CKI-1::GFP localization. Additional RNAi experiments revealed that the SCF E3 ubiquitin-ligase complex genes, skpt-1/SKP2, cul-1/CUL1 and lin-23/F-bo...
In C. elegans, oocytes are ovulated into the spermatheca, where they are fertilized before being ... more In C. elegans, oocytes are ovulated into the spermatheca, where they are fertilized before being pushed into the uterus. Contraction in the C. elegans spermatheca is driven by circumferential acto-myosin fibers. The C. elegans zyxin homolog, zyx-1, is expressed in the body wall muscle, pharynx and spermatheca. To our surprise, a CRISPR-generated zyx-1 deletion allele results in no overt developmental phenotypes, and the spermathecal actin cytoskeleton appears wild type, however, oocyte transit through the spermatheca is slower than in wild type animals. This suggests ZYX-1/Zyxin may regulate spermathecal contraction magnitude or timing of spermathecal bag contraction and/or spermathecal-uterine valve dilation.
Biochemical and Biophysical Research Communications, 2022
UNC-52/perlecan is a basement membrane (BM) proteoglycan playing an essential role in the muscle ... more UNC-52/perlecan is a basement membrane (BM) proteoglycan playing an essential role in the muscle cell attachment of C. elegans. The UNC-52 protein contains two RGD (Arg-Gly-Asp) motifs in domains III and IV, a well-characterized tripeptide known for binding to mammalian β integrin. To investigate the role of the RGD motif in UNC-52/perlecan, we created two mutations in the 2021RGD2023 motif: one mutation changed the RGD to an RGE, and the other deleted the RGD motif. The RGE2023 caused defective actin filaments and aberrant localization of PAT-3 β integrin and TLN-1/talin. Additionally, the in-frame deletion of RGD2023 resulted in a paralyzed and arrested at two-fold embryonic stages (Pat) phenotype, which is the identical phenotype of the pat-3 β integrin null allele. These results indicate that RGD2023 is a potential ligand for cell binding and is essential for development and survival. Furthermore, our analysis reveals that the RGD of an invertebrate BM molecule is a potential cell-binding motif, suggesting that the function of the RGD motif is conserved among species.
Figure 1: Characterization of PAT-3 membrane distal NPxY phospho-tyrosine motif. (A) N2 hermaphro... more Figure 1: Characterization of PAT-3 membrane distal NPxY phospho-tyrosine motif. (A) N2 hermaphrodite gonad. Arrowhead and path indicate distal tip cell (DTC) migration. Bar = 100 μm; (B) A pat-3(kq8041), Y804A, gonad showing migration defect. The gonad arm made extra turns. Arrowhead and path indicate DTC migration. Bar = 50 μm; (C) Protein sequence of wild type and mutant PAT-3 cytoplasmic tail was compared to human β1 integrin. Reds are the tyrosine and mutant residues in membrane distal NPxY; D. Gonad migration and motility analyses of pat-3 mutants.
Figure 1. Wild-type N2 strain of C. elegans was stimulated by Salmonella enterica lipopolysacchar... more Figure 1. Wild-type N2 strain of C. elegans was stimulated by Salmonella enterica lipopolysaccharide (LPS) to lay eggs in an aqueous environment: Animals were incubated in M9 buffer for 15 minutes, after which the numbers of eggs laid were recorded for adjustment purpose. The liquid medium was then supplemented with either LPS or serotonin, or with no supplement as the negative control. The final concentration of LPS was 0.1 mg/ml and that of serotonin was 1 mg/ml. After one hour, the eggs in each microtiter well were counted again. For each individual, the adjusted number of eggs laid, which considered only the eggs laid during the treatment period, is presented here. More than 100 animals under each condition were tested. Error bars represent standard errors; asterisks denotes p < 0.01 when the treatment condition (LPS or serotonin) resulted in an egg-laying level that was significantly different from that of the negative control condition (M9), when the non-parametric Mann-Whi...
Figure 1: A: Muscle cell of NK358 pat-3::GFP animal. The dotted lines represent dense bodies (arr... more Figure 1: A: Muscle cell of NK358 pat-3::GFP animal. The dotted lines represent dense bodies (arrows) and straight lines represent M-lines (arrowheads); B: Muscle cell of a pat-3::GFP; unc-52(kq748) animal. The dotted lines represent dense bodies (arrows) and straight lines (arrowheads) represent M-lines. Localization appears similar to Figure 1A; C: Rhodamine-conjugated phalloidin staining of an N2 muscle cell. Actin cytoskeletons along the length of muscle are stained (arrows); D: Rhodamine-conjugated phalloidin staining of an unc-52(kq748) muscle cell. No obvious abnormalities in thin (actin) filaments (arrows) are present. Scale bar = 10 μm.; E: Thrashing assay results for unc-52 (kq748) (1.4454 average thrashes/second, n=50), unc-52(kq745) (1.339 average thrashes/second, n=50), and N2 wild-type (1.99 average thrashes/second, n=50). * p-value < 0.05 compared to N2 wild-type.
The lon-2 gene in Caenorhabditis elegans encodes a heparan sulfate proteoglycan family glypican t... more The lon-2 gene in Caenorhabditis elegans encodes a heparan sulfate proteoglycan family glypican that negatively regulates the BMP signaling pathway responsible for controlling body length. LON-2 contains multiple functional domains, including an RGD (Arg-Gly-Asp) motif at amino acid number from 348 to 350. A novel mutant allele of lon-2 was investigated in this study. In this mutant allele, lon-2(kq348ΔRGD), the RGD motif at position 348 was deleted. Another pre-existing mutant allele, lon-2(e678), contains a ~9kb deletion and lacks most of the genomic coding sequence. The lon-2(e678) line was used as a reference allele. The novel mutant line was significantly shorter than wild-type animals, suggesting that removal of the RGD motif in LON-2 may improve its ability to inhibit BMP signaling.
The cell-extracellular matrix (ECM) interaction plays an essential role in maintaining tissue sha... more The cell-extracellular matrix (ECM) interaction plays an essential role in maintaining tissue shapes and regulates cell behaviors such as cell adhesion, differentiation and proliferation. The mechanism by which the ECM influences the cell cycle in vivo is poorly understood. Here we demonstrate that the β integrin PAT-3 regulates the localization and expression of CKI-1, a C. elegans homologue of the cyclin dependent kinase inhibitor p27(KIP1). In nematodes expressing wild type PAT-3, CKI-1::GFP localizes primarily to nucleoli in hypodermal cells, whereas in animals expressing mutant pat-3 with a defective splice junction, CKI-1::GFP appears clumped and disorganized in nucleoplasm. RNAi analysis links cell adhesion genes to the regulation of CKI-1. RNAi of unc-52/perlecan, ina-1/α integrin, pat-4/ILK, and unc-97/PINCH resulted in abnormal CKI-1::GFP localization. Additional RNAi experiments revealed that the SCF E3 ubiquitin-ligase complex genes, skpt-1/SKP2, cul-1/CUL1 and lin-23/F-...
Correct distal tip cell (DTC) migration in the nematode C. elegans requires sensing soluble and m... more Correct distal tip cell (DTC) migration in the nematode C. elegans requires sensing soluble and matrix cues, remodeling extracellular matrix, and signaling through conserved integrin and netrin pathways. The DTC executes a complex path and coordinates its migration with the developmental stages of larval morphogenesis. This chapter outlines a method for investigating DTC migration in C. elegans using feeding RNA interference (RNAi) and light microscopy. To deplete a candidate gene of interest, nematode eggs are added to plates seeded with RNAi-inducing bacterial lawns. The animals hatch and begin to eat the RNAi bacteria, releasing dsRNA and causing the targeted gene to be depleted during larval development. Positions of migratory cells are monitored in larvae and young adults using differential interference contrast (DIC) and epifluorescence microscopy.
Tissue morphogenesis requires proper interaction between cells and the extracellular matrix (ECM)... more Tissue morphogenesis requires proper interaction between cells and the extracellular matrix (ECM), which is mediated by alphabeta heterodimeric receptor integrin. In Caenorhabditis elegans, integrin signaling is essential for formation of gonad. Here, we probe the role of several integrin-associated molecules in ovulation and cell migration. Function of pat-4/integrin-linked kinase (ILK) and unc-112/Mig-2 was examined using RNA-mediated interference (RNAi). Depletion of these messages caused oocyte accumulation in the proximal gonad and distal tip cells (DTC) migration defects. It was further determined that failed ovulation was due to defective contraction and dilation of somatic gonad structures, including spermatheca and gonad sheath. Actin cytoskeleton in the proximal gonad of RNAi animals appeared disorganized, indicating that RNAi of pat-4 or unc-112 inhibited the overall assembly of actin cytoskeleton in somatic gonad. Taken together, our analysis confirms the role of integrin and integrin-associated proteins in gonad function.
TLN-1/talin is a conserved focal adhesion protein that forms part of the linkage between the cyto... more TLN-1/talin is a conserved focal adhesion protein that forms part of the linkage between the cytoplasmic tail of integrin and the actin cytoskeleton. In C. elegans , TLN-1 is expressed strongly in striated muscle and the gonadal sheath cells. Here, we report that a CRISPR-generated TLN-1 allele TLN-1(W387A), predicted to affect binding of talin to integrins, results in mild phenotypes, including motility defects and ovulation defects. The arrangement of the actin cytoskeleton in the body wall muscles, spermatheca, and sheath appears identical in wild type and TLN-1(W387A) animals. This analysis suggests that W387 in TLN-1 does not have a major effect on the binding of talin to integrin in vivo .
<p>Panel A: CKI-1::GFP expression levels were assessed in the transgenic rescued lines. Top... more <p>Panel A: CKI-1::GFP expression levels were assessed in the transgenic rescued lines. Top bands in lanes 1 and 2 show the relative level of UNC-54/myosin B in each sample. Bottom bands indicate the level of CKI-1::GFP. Lanes 1 and 2 represent <i>pat-3</i>(+) and <i>pat-3(sp)</i>, respectively. Quantification (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042425#pone.0042425.s003" target="_blank">Table S1</a>) using ImageJ software revealed that CKI-1::GFP level was 10-fold increased in <i>pat-3(sp)</i> animals compared to <i>pat-3</i>(+) animals. Panel B: CKI-1::GFP expression levels were assessed in <i>pat-3</i>(+) animals treated with RNAi directed against focal adhesion genes. Top bands represent the amount of CKI-1::GFP in extracts prepared from each RNAi condition. L4440 is a negative RNAi control. The <i>pat-3</i>, <i>ina-1</i>, <i>unc-97</i>, <i>unc-52, pat-6</i> and <i>let-2</i> RNAi caused upregulation of CKI-1::GFP, while <i>unc-112</i> RNAi had no effect. Bottom bands indicate MH33 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042425#pone.0042425-Francis1" target="_blank">[90]</a> levels in each lane as a loading control. Quantification (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042425#pone.0042425.s003" target="_blank">Table S1</a>) using ImageJ software revealed that CKI-1::GFP level was increased by RNAi of <i>pat-3</i>, <i>ina-1</i>, <i>unc-97</i>, <i>unc-52, pat-6,</i> and <i>let-2</i>. Panel C: CKI-1::GFP expression levels were also measured in <i>pat-3</i>(+) animals treated with E3 ligase gene RNAi. Top bands represent the amount of CKI-1::GFP in the extracts prepared from each RNAi condition. L4440 is a negative RNAi control. The <i>skpt-1</i>, <i>lin-23</i> and <i>cul-1</i> RNAi depletions caused upregulation of CKI-1::GFP, while <i>rbx-1</i> and <i>cul-4</i> RNAi had no effect. Bottom bands indicate MH33 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042425#pone.0042425-Francis1" target="_blank">[90]</a> levels in each lane as a loading control. Quantification (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042425#pone.0042425.s003" target="_blank">Table S1</a>) using ImageJ software revealed that the CKI-1::GFP level was increased by RNAi of <i>skpt-1</i>, <i>lin-23</i> and <i>cul-1</i>.</p
The cell-extracellular matrix (ECM) interaction plays an essential role in maintaining tissue sha... more The cell-extracellular matrix (ECM) interaction plays an essential role in maintaining tissue shapes and regulates cell behaviors such as cell adhesion, differentiation and proliferation. The mechanism by which the ECM influences the cell cycle in vivo is poorly understood. Here we demonstrate that the b integrin PAT-3 regulates the localization and expression of CKI-1, a C. elegans homologue of the cyclin dependent kinase inhibitor p27KIP1. In nematodes expressing wild type PAT-3, CKI-1::GFP localizes primarily to nucleoli in hypodermal cells, whereas in animals expressing mutant pat-3 with a defective splice junction, CKI-1::GFP appears clumped and disorganized in nucleoplasm. RNAi analysis links cell adhesion genes to the regulation of CKI-1. RNAi of unc-52/perlecan, ina-1/a integrin, pat-4/ILK, and unc-97/PINCH resulted in abnormal CKI-1::GFP localization. Additional RNAi experiments revealed that the SCF E3 ubiquitin-ligase complex genes, skpt-1/SKP2, cul-1/CUL1 and lin-23/F-bo...
In C. elegans, oocytes are ovulated into the spermatheca, where they are fertilized before being ... more In C. elegans, oocytes are ovulated into the spermatheca, where they are fertilized before being pushed into the uterus. Contraction in the C. elegans spermatheca is driven by circumferential acto-myosin fibers. The C. elegans zyxin homolog, zyx-1, is expressed in the body wall muscle, pharynx and spermatheca. To our surprise, a CRISPR-generated zyx-1 deletion allele results in no overt developmental phenotypes, and the spermathecal actin cytoskeleton appears wild type, however, oocyte transit through the spermatheca is slower than in wild type animals. This suggests ZYX-1/Zyxin may regulate spermathecal contraction magnitude or timing of spermathecal bag contraction and/or spermathecal-uterine valve dilation.
Biochemical and Biophysical Research Communications, 2022
UNC-52/perlecan is a basement membrane (BM) proteoglycan playing an essential role in the muscle ... more UNC-52/perlecan is a basement membrane (BM) proteoglycan playing an essential role in the muscle cell attachment of C. elegans. The UNC-52 protein contains two RGD (Arg-Gly-Asp) motifs in domains III and IV, a well-characterized tripeptide known for binding to mammalian β integrin. To investigate the role of the RGD motif in UNC-52/perlecan, we created two mutations in the 2021RGD2023 motif: one mutation changed the RGD to an RGE, and the other deleted the RGD motif. The RGE2023 caused defective actin filaments and aberrant localization of PAT-3 β integrin and TLN-1/talin. Additionally, the in-frame deletion of RGD2023 resulted in a paralyzed and arrested at two-fold embryonic stages (Pat) phenotype, which is the identical phenotype of the pat-3 β integrin null allele. These results indicate that RGD2023 is a potential ligand for cell binding and is essential for development and survival. Furthermore, our analysis reveals that the RGD of an invertebrate BM molecule is a potential cell-binding motif, suggesting that the function of the RGD motif is conserved among species.
Figure 1: Characterization of PAT-3 membrane distal NPxY phospho-tyrosine motif. (A) N2 hermaphro... more Figure 1: Characterization of PAT-3 membrane distal NPxY phospho-tyrosine motif. (A) N2 hermaphrodite gonad. Arrowhead and path indicate distal tip cell (DTC) migration. Bar = 100 μm; (B) A pat-3(kq8041), Y804A, gonad showing migration defect. The gonad arm made extra turns. Arrowhead and path indicate DTC migration. Bar = 50 μm; (C) Protein sequence of wild type and mutant PAT-3 cytoplasmic tail was compared to human β1 integrin. Reds are the tyrosine and mutant residues in membrane distal NPxY; D. Gonad migration and motility analyses of pat-3 mutants.
Figure 1. Wild-type N2 strain of C. elegans was stimulated by Salmonella enterica lipopolysacchar... more Figure 1. Wild-type N2 strain of C. elegans was stimulated by Salmonella enterica lipopolysaccharide (LPS) to lay eggs in an aqueous environment: Animals were incubated in M9 buffer for 15 minutes, after which the numbers of eggs laid were recorded for adjustment purpose. The liquid medium was then supplemented with either LPS or serotonin, or with no supplement as the negative control. The final concentration of LPS was 0.1 mg/ml and that of serotonin was 1 mg/ml. After one hour, the eggs in each microtiter well were counted again. For each individual, the adjusted number of eggs laid, which considered only the eggs laid during the treatment period, is presented here. More than 100 animals under each condition were tested. Error bars represent standard errors; asterisks denotes p < 0.01 when the treatment condition (LPS or serotonin) resulted in an egg-laying level that was significantly different from that of the negative control condition (M9), when the non-parametric Mann-Whi...
Figure 1: A: Muscle cell of NK358 pat-3::GFP animal. The dotted lines represent dense bodies (arr... more Figure 1: A: Muscle cell of NK358 pat-3::GFP animal. The dotted lines represent dense bodies (arrows) and straight lines represent M-lines (arrowheads); B: Muscle cell of a pat-3::GFP; unc-52(kq748) animal. The dotted lines represent dense bodies (arrows) and straight lines (arrowheads) represent M-lines. Localization appears similar to Figure 1A; C: Rhodamine-conjugated phalloidin staining of an N2 muscle cell. Actin cytoskeletons along the length of muscle are stained (arrows); D: Rhodamine-conjugated phalloidin staining of an unc-52(kq748) muscle cell. No obvious abnormalities in thin (actin) filaments (arrows) are present. Scale bar = 10 μm.; E: Thrashing assay results for unc-52 (kq748) (1.4454 average thrashes/second, n=50), unc-52(kq745) (1.339 average thrashes/second, n=50), and N2 wild-type (1.99 average thrashes/second, n=50). * p-value < 0.05 compared to N2 wild-type.
The lon-2 gene in Caenorhabditis elegans encodes a heparan sulfate proteoglycan family glypican t... more The lon-2 gene in Caenorhabditis elegans encodes a heparan sulfate proteoglycan family glypican that negatively regulates the BMP signaling pathway responsible for controlling body length. LON-2 contains multiple functional domains, including an RGD (Arg-Gly-Asp) motif at amino acid number from 348 to 350. A novel mutant allele of lon-2 was investigated in this study. In this mutant allele, lon-2(kq348ΔRGD), the RGD motif at position 348 was deleted. Another pre-existing mutant allele, lon-2(e678), contains a ~9kb deletion and lacks most of the genomic coding sequence. The lon-2(e678) line was used as a reference allele. The novel mutant line was significantly shorter than wild-type animals, suggesting that removal of the RGD motif in LON-2 may improve its ability to inhibit BMP signaling.
The cell-extracellular matrix (ECM) interaction plays an essential role in maintaining tissue sha... more The cell-extracellular matrix (ECM) interaction plays an essential role in maintaining tissue shapes and regulates cell behaviors such as cell adhesion, differentiation and proliferation. The mechanism by which the ECM influences the cell cycle in vivo is poorly understood. Here we demonstrate that the β integrin PAT-3 regulates the localization and expression of CKI-1, a C. elegans homologue of the cyclin dependent kinase inhibitor p27(KIP1). In nematodes expressing wild type PAT-3, CKI-1::GFP localizes primarily to nucleoli in hypodermal cells, whereas in animals expressing mutant pat-3 with a defective splice junction, CKI-1::GFP appears clumped and disorganized in nucleoplasm. RNAi analysis links cell adhesion genes to the regulation of CKI-1. RNAi of unc-52/perlecan, ina-1/α integrin, pat-4/ILK, and unc-97/PINCH resulted in abnormal CKI-1::GFP localization. Additional RNAi experiments revealed that the SCF E3 ubiquitin-ligase complex genes, skpt-1/SKP2, cul-1/CUL1 and lin-23/F-...
Correct distal tip cell (DTC) migration in the nematode C. elegans requires sensing soluble and m... more Correct distal tip cell (DTC) migration in the nematode C. elegans requires sensing soluble and matrix cues, remodeling extracellular matrix, and signaling through conserved integrin and netrin pathways. The DTC executes a complex path and coordinates its migration with the developmental stages of larval morphogenesis. This chapter outlines a method for investigating DTC migration in C. elegans using feeding RNA interference (RNAi) and light microscopy. To deplete a candidate gene of interest, nematode eggs are added to plates seeded with RNAi-inducing bacterial lawns. The animals hatch and begin to eat the RNAi bacteria, releasing dsRNA and causing the targeted gene to be depleted during larval development. Positions of migratory cells are monitored in larvae and young adults using differential interference contrast (DIC) and epifluorescence microscopy.
Tissue morphogenesis requires proper interaction between cells and the extracellular matrix (ECM)... more Tissue morphogenesis requires proper interaction between cells and the extracellular matrix (ECM), which is mediated by alphabeta heterodimeric receptor integrin. In Caenorhabditis elegans, integrin signaling is essential for formation of gonad. Here, we probe the role of several integrin-associated molecules in ovulation and cell migration. Function of pat-4/integrin-linked kinase (ILK) and unc-112/Mig-2 was examined using RNA-mediated interference (RNAi). Depletion of these messages caused oocyte accumulation in the proximal gonad and distal tip cells (DTC) migration defects. It was further determined that failed ovulation was due to defective contraction and dilation of somatic gonad structures, including spermatheca and gonad sheath. Actin cytoskeleton in the proximal gonad of RNAi animals appeared disorganized, indicating that RNAi of pat-4 or unc-112 inhibited the overall assembly of actin cytoskeleton in somatic gonad. Taken together, our analysis confirms the role of integrin and integrin-associated proteins in gonad function.
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Papers by Myeongwoo Lee