scholarly journals Drosophila Kelch functions with Cullin-3 to organize the ring canal actin cytoskeleton

2010 ◽  
Vol 188 (1) ◽  
pp. 29-37 ◽  
Author(s):  
Andrew M. Hudson ◽  
Lynn Cooley
Keyword(s):  
Repeated Sequence ◽  
Adaptor Proteins ◽  
Sequence Motif ◽  
Filamentous Actin ◽  
E3 Ligases ◽  
Cytoskeletal Structure ◽  
Ring Canals ◽  
Cullin 3 ◽  
New Gene ◽  
Female Germline

Drosophila melanogaster Kelch (KEL) is the founding member of a diverse protein family defined by a repeated sequence motif known as the KEL repeat (KREP). Several KREP proteins, including Drosophila KEL, bind filamentous actin (F-actin) and contribute to its organization. Recently, a subset of KREP proteins has been shown to function as substrate adaptor proteins for cullin-RING (really interesting new gene) ubiquitin E3 ligases. In this study, we demonstrate that association of Drosophila KEL with Cullin-3, likely in a cullin-RING ligase, is essential for the growth of Drosophila female germline ring canals. These results suggest a role for protein ubiquitylation in the remodeling of a complex F-actin cytoskeletal structure.

scholarly journals Targeted substrate degradation by Kelch controls the actin cytoskeleton during ring canal expansion

2018 ◽  
Author(s):  
Andrew M. Hudson ◽  
Katelynn M. Mannix ◽  
Julianne A. Gerdes ◽  
Molly C. Kottemann ◽  
Lynn Cooley
Keyword(s):  
Actin Cytoskeleton ◽  
Affinity Purification ◽  
Ubiquitin Proteasome System ◽  
Sequence Motif ◽  
Ring Canal ◽  
Cytoskeletal Structure ◽  
Ubiquitin Ligase Complex ◽  
Ring Canals ◽  
Cullin 3 ◽  
Ubiquitin Proteasome

AbstractDuring Drosophila oogenesis, specialized actin-based structures called ring canals form and expand to accommodate growth of the oocyte. Previous work demonstrated that Kelch and Cullin 3 function together in a Cullin 3-RING ubiquitin ligase complex (CRL3Kelch) to organize the ring canal cytoskeleton, presumably by targeting a substrate for proteolysis. Here, we use tandem affinity purification followed by mass spectrometry to identify HtsRC as the CRL3Kelch ring canal substrate. CRISPR-mediated mutagenesis of HtsRC revealed its requirement in the recruitment of the ring canal F-actin cytoskeleton. We present genetic evidence consistent with HtsRC being the CRL3Kelch substrate, as well as biochemical evidence indicating that HtsRC is ubiquitylated and degraded by the proteasome. Finally, we identify a short sequence motif in HtsRC that is necessary for Kelch binding. These findings uncover an unusual mechanism during development wherein a specialized cytoskeletal structure is regulated and remodeled by the ubiquitin-proteasome system.

scholarly journals HtsRC-mediated accumulation of F-actin regulates ring canal size during Drosophila melanogaster oogenesis

2020 ◽  
Author(s):  
Julianne A. Gerdes ◽  
Katelynn M. Mannix ◽  
Andrew M. Hudson ◽  
Lynn Cooley
Keyword(s):  
Drosophila Melanogaster ◽  
Actin Cytoskeleton ◽  
Fruit Flies ◽  
Follicle Cells ◽  
Filamentous Actin ◽  
Ring Canal ◽  
High Fecundity ◽  
Ring Canals ◽  
Necessary And Sufficient ◽  
Female Germline

AbstractRing canals in the female germline of Drosophila melanogaster are supported by a robust filamentous actin (F-actin) cytoskeleton, setting them apart from ring canals in other species and tissues. Previous work has identified components required for the expansion of the ring canal actin cytoskeleton but has not identified the proteins responsible for F-actin recruitment or accumulation. Using a combination of CRISPR-Cas9 mediated mutagenesis and UAS-Gal4 overexpression, we show that HtsRC, a component specific to female germline ring canals, is both necessary and sufficient to drive F-actin accumulation. Absence of HtsRC in the germline resulted in ring canals lacking inner rim F-actin, while overexpression of HtsRC led to larger ring canals. HtsRC functions in combination with Filamin to recruit F-actin to ring-canal-like ectopic actin structures in somatic follicle cells. Finally, we present findings which indicate that HtsRC expression and robust female germline ring canal expansion are important for high fecundity in fruit flies but dispensable for their fertility, a result which is consistent with our understanding of HtsRC as a newly evolved gene specific to female germline ring canals.

scholarly journals HtsRC-Mediated Accumulation of F-Actin Regulates Ring Canal Size During Drosophila melanogaster Oogenesis

Genetics ◽  
2020 ◽  
Vol 216 (3) ◽  
pp. 717-734
Author(s):  
Julianne A. Gerdes ◽  
Katelynn M. Mannix ◽  
Andrew M. Hudson ◽  
Lynn Cooley
Keyword(s):  
Drosophila Melanogaster ◽  
Actin Cytoskeleton ◽  
Fruit Flies ◽  
Follicle Cells ◽  
Filamentous Actin ◽  
Ring Canal ◽  
High Fecundity ◽  
Ring Canals ◽  
Necessary And Sufficient ◽  
Female Germline

Ring canals in the female germline of Drosophila melanogaster are supported by a robust filamentous actin (F-actin) cytoskeleton, setting them apart from ring canals in other species and tissues. Previous work has identified components required for the expansion of the ring canal actin cytoskeleton, but has not identified the proteins responsible for F-actin recruitment or accumulation. Using a combination of CRISPR-Cas9 mediated mutagenesis and UAS-Gal4 overexpression, we show that HtsRC—a component specific to female germline ring canals—is both necessary and sufficient to drive F-actin accumulation. Absence of HtsRC in the germline resulted in ring canals lacking inner rim F-actin, while overexpression of HtsRC led to larger ring canals. HtsRC functions in combination with Filamin to recruit F-actin to ectopic actin structures in somatic follicle cells. Finally, we present findings that indicate that HtsRC expression and robust female germline ring canal expansion are important for high fecundity in fruit flies but dispensable for their fertility—a result that is consistent with our understanding of HtsRC as a newly evolved gene specific to female germline ring canals.

scholarly journals Formation of the Drosophila Ovarian Ring Canal Inner Rim Depends on cheerio

Genetics ◽  
1997 ◽  
Vol 145 (4) ◽  
pp. 1063-1072 ◽  
Author(s):  
Douglas N Robinson ◽  
Tracy A Smith-Leiker ◽  
Nicholas S Sokol ◽  
Andrew M Hudson ◽  
Lynn Cooley
Keyword(s):  
Mature Oocyte ◽  
Cleavage Furrow ◽  
Intercellular Bridge ◽  
Filamentous Actin ◽  
Ring Canal ◽  
Membrane Stabilization ◽  
Critical Function ◽  
Ring Canals ◽  
New Gene ◽  
Mutant Cells

In Drosophila oogenesis, the development of a mature oocyte depends on having properly developed ring canals that allow cytoplasm transport from the nurse cells to the oocyte. Ring canal assembly is a step-wise process that transforms an arrested cleavage furrow into a stable intercellular bridge by the addition of several proteins. Here we describe a new gene we named cheerio that provides a critical function for ring canal assembly. Mutants in cheerio fail to localize ring canal inner rim proteins including filamentous actin, the ring canal-associated products from the hu-li tai shao (hts) gene, and kelch. Since hts and kelch are present but unlocalized in cheerio mutant cells, cheerio is likely to function upstream from each of them. Examination of mutants in cheerio places it in the pathway of ring canal assembly between cleavage furrow arrest and localization of hts and actin filaments. Furthermore, this mutant reveals that the inner rim cytoskeleton is required for expansion of the ring canal opening and for plasma membrane stabilization.

Targeting gene expression to the head: the Drosophila orthodenticle gene is a direct target of the Bicoid morphogen

Development ◽  
1998 ◽  
Vol 125 (21) ◽  
pp. 4185-4193 ◽  
Author(s):  
Q. Gao ◽  
R. Finkelstein
Keyword(s):  
Gene Expression ◽  
Target Genes ◽  
Regulatory Element ◽  
Regulatory Region ◽  
Repeated Sequence ◽  
Drosophila Embryo ◽  
Sequence Motif ◽  
Specific Expression ◽  
Gap Gene

The Bicoid (Bcd) morphogen establishes the head and thorax of the Drosophila embryo. Bcd activates the transcription of identified target genes in the thoracic segments, but its mechanism of action in the head remains poorly understood. It has been proposed that Bcd directly activates the cephalic gap genes, which are the first zygotic genes to be expressed in the head primordium. It has also been suggested that the affinity of Bcd-binding sites in the promoters of Bcd target genes determines the posterior extent of their expression (the Gene X model). However, both these hypotheses remain untested. Here, we show that a small regulatory region upstream of the cephalic gap gene orthodenticle (otd) is sufficient to recapitulate early otd expression in the head primordium. This region contains two control elements, each capable of driving otd-like expression. The first element has consensus Bcd target sites that bind Bcd in vitro and are necessary for head-specific expression. As predicted by the Gene X model, this element has a relatively low affinity for Bcd. Surprisingly, the second regulatory element has no Bcd sites. Instead, it contains a repeated sequence motif similar to a regulatory element found in the promoters of otd-related genes in vertebrates. Our study is the first demonstration that a cephalic gap gene is directly regulated by Bcd. However, it also shows that zygotic gene expression can be targeted to the head primordium without direct Bcd regulation.

Morphogenesis of Drosophila ovarian ring canals

Development ◽  
1994 ◽  
Vol 120 (7) ◽  
pp. 2015-2025 ◽  
Author(s):  
D.N. Robinson ◽  
K. Cant ◽  
L. Cooley
Keyword(s):  
Protein Assembly ◽  
Wild Type ◽  
Filamentous Actin ◽  
Ring Canal ◽  
Ring Canals ◽  
Normal Ring ◽  
Egg Chambers ◽  
Cytoplasmic Bridges ◽  
Carboxy Terminal ◽  
Ordered Ring

We analyzed the structure of cytoplasmic bridges called ring canals in Drosophila egg chambers. Two mutations, hu-li tai shao (hts) and kelch, disrupt normal ring canal development. We raised antibodies against the carboxy-terminal tail of hts and found that they recognize a protein that localizes specifically to ring canals very early in ring canal assembly. Accumulation of filamentous actin on ring canals coincides with the appearance of the hts protein. kelch, which is localized to the ring canals hours after hts and actin, is necessary for maintaining a highly ordered ring canal rim since kelch mutant egg chambers have ring canals that are obstructed by disordered actin and hts. Anti-phosphotyrosine antibodies immunostain ring canals beginning early in the germarium before hts and actin and throughout egg chamber development. The use of antibody reagents to analyze the structure of wild-type and mutant ring canals has shown that ring canal development is a dynamic process of cytoskeletal protein assembly, possibly regulated by tyrosine phosphorylation of some ring canal components.

scholarly journals Iterons Homologous to Helper Geminiviruses Are Essential for Efficient Replication of Betasatellites

2018 ◽  
Vol 93 (5) ◽  
Author(s):  
Xiongbiao Xu ◽  
Yajuan Qian ◽  
Yaqin Wang ◽  
Zhenghe Li ◽  
Xueping Zhou
Keyword(s):  
Tandem Repeats ◽  
Repeated Sequence ◽  
Iterative Sequence ◽  
Binding Motif ◽  
Sequence Motif ◽  
High Affinity ◽  
Efficient Replication ◽  
Tobacco Curly Shoot Virus ◽  
Species Specific ◽  
Origin Recognition

ABSTRACTBetasatellites associated with geminiviruses can be replicated promiscuously by distinct geminiviruses but exhibit a preference for cognate helper viruses. However, theciselements responsible for betasatellite origin recognition have not been characterized. In this study, we identified an iteron-like repeated sequence motif, 5′-GAGGACC-3′, in a tobacco curly shoot betasatellite (TbCSB) associated with tobacco curly shoot virus (TbCSV). Competitive DNA binding assays revealed that two core repeats (5′-GGACC-3′) are required for specific binding to TbCSV Rep; TbCSB iteron mutants accumulated to greatly reduced levels and lost the cognate helper-mediated replication preference. Interestingly, TbCSV also contains identical repeated sequences that are essential for specific Rep binding andin vivoreplication. In order to gain insight into the mechanism by which TbCSB has acquired the cognate iterons, we performed a SELEX (systematic evolution of ligands by exponential enrichment) assay to identify the high-affinity Rep binding ligands from a large pool of randomized sequences. Analysis of SELEX winners showed that all of the sequences contained at least one core iteron-like motif, suggesting that TbCSB has evolved to contain cognate iterons for high-affinity Rep binding. Further analyses of various betasatellite sequences revealed a region upstream of the satellite conserved region replete with iterative sequence motifs, including species-specific repeats and a general repeat (5′-GGTAAAT-3′). Remarkably, the species-specific repeats in many betasatellites are homologous to those in their respective cognate helper begomoviruses, whereas the general repeat is widespread in most of the betasatellite molecules analyzed. These data, taken together, suggest that many betasatellites have evolved to acquire homologous iteron-like sequences for efficient replication mediated by cognate helper viruses.IMPORTANCEThe geminivirus-encoded replication initiator protein (Rep) binds to repeated sequence elements (also known as iterons) in the origin of replication that serve as essentialciselements for specific viral replication. Betasatellites associated with begomoviruses can be replicated by cognate or noncognate helper viruses, but theciselements responsible for betasatellite origin recognition have not been characterized. Using a betasatellite (TbCSB) associated with tobacco curly shoot virus (TbCSV) as a model, we identify two tandem repeats (iterons) in the Rep-binding motif (RBM) that are required for specific Rep binding and efficient replication, and we show that identical iteron sequences present in TbCSV are also necessary for Rep binding and the replication of helper viruses. Extensive analysis of begomovirus/betasatellite sequences shows that many betasatellites contain iteron-like elements homologous to those of their respective cognate helper begomoviruses. Our data suggest that many betasatellites have evolved to acquire homologous iteron-like sequences for efficient replication mediated by cognate helper viruses.

scholarly journals Brd4 Regulation of Papillomavirus Protein E2 Stability

2009 ◽  
Vol 83 (17) ◽  
pp. 8683-8692 ◽  
Author(s):  
Gang Zheng ◽  
Michal-Ruth Schweiger ◽  
Gustavo Martinez-Noel ◽  
Leon Zheng ◽  
Jennifer A. Smith ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Activation Function ◽  
Dominant Negative ◽  
Genome Maintenance ◽  
E2 Protein ◽  
E3 Ligases ◽  
Protein Levels ◽  
Dividing Cells ◽  
Cullin 3 ◽  
Infected Cells

ABSTRACT The papillomavirus (PV) E2 protein is an important regulator of the viral life cycle. It has diverse roles in viral transcription, DNA replication, and genome maintenance. Our laboratory has previously identified the cellular bromodomain protein Brd4 as a key interacting partner of E2. Brd4 mediates the transcriptional activation function of E2 and plays an important role in viral genome maintenance in dividing cells. E2 interacts with the C-terminal domain (CTD) of Brd4, and the CTD functions in a dominant-negative manner through binding E2 and interfering with E2's interaction with the full-length Brd4 protein. Previous studies have shown that PV E2 proteins are short lived; however, the mechanisms regulating their stability and degradation have not yet been well established. In this study, we explored the role of Brd4 in the regulation of bovine PV 1 (BPV1) and human PV 16 (HPV16) E2 stability. Expression of the Brd4 CTD dramatically increases E2 levels. Both BPV1 E2 and HPV16 E2 are regulated by ubiquitylation, and Brd4 CTD expression blocks this ubiquitylation, thus stabilizing the E2 protein. Furthermore, we have identified the cullin-based E3 ligases and specifically cullin-3 as potential components of the ubiquitylation machinery that targets both BPV1 and HPV16 E2 for ubiquitylation. Expression of the Brd4 CTD blocks the interaction between E2 and the cullin-3 complex. In addition to Brd4's role in mediating E2 transcription and genome tethering activities, these data suggest a potential role for Brd4 in regulating E2 stability and protein levels within PV-infected cells.

scholarly journals Cdc42hs Facilitates Cytoskeletal Reorganization and Neurite Outgrowth by Localizing the 58-Kd Insulin Receptor Substrate to Filamentous Actin

2001 ◽  
Vol 152 (3) ◽  
pp. 579-594 ◽  
Author(s):  
Sheila Govind ◽  
Robert Kozma ◽  
Clinton Monfries ◽  
Louis Lim ◽  
Sohail Ahmed
Keyword(s):  
Insulin Receptor ◽  
Binding Site ◽  
Neurite Outgrowth ◽  
Adaptor Proteins ◽  
Cytoskeletal Reorganization ◽  
Filamentous Actin ◽  
3T3 Cells ◽  
Insulin Receptor Tyrosine Kinase ◽  
Interaction Sites ◽  
Swiss 3T3

Cdc42Hs is involved in cytoskeletal reorganization and is required for neurite outgrowth in N1E-115 cells. To investigate the molecular mechanism by which Cdc42Hs regulates these processes, a search for novel Cdc42Hs protein partners was undertaken by yeast two-hybrid assay. Here, we identify the 58-kD substrate of the insulin receptor tyrosine kinase (IRS-58) as a Cdc42Hs target. IRS-58 is a brain-enriched protein comprising at least four protein–protein interaction sites: a Cdc42Hs binding site, an Src homology (SH)3-binding site, an SH3 domain, and a tryptophan, tyrptophan (WW)-binding domain. Expression of IRS-58 in Swiss 3T3 cells leads to reorganization of the filamentous (F)-actin cytoskeleton, involving loss of stress fibers and formation of filopodia and clusters. In N1E-115 cells IRS-58 induces neurite outgrowth with high complexity. Expression of a deletion mutant of IRS-58, which lacks the SH3- and WW-binding domains, induced neurite extension without complexity in N1E-115 cells. In Swiss 3T3 cells and N1E-115 cells, IRS-58 colocalizes with F-actin in clusters and filopodia. An IRS-581267N mutant unable to bind Cdc42Hs failed to localize with F-actin to induce neurite outgrowth or significant cytoskeletal reorganization. These results suggest that Cdc42Hs facilitates cytoskeletal reorganization and neurite outgrowth by localizing protein complexes via adaptor proteins such as IRS-58 to F-actin.

scholarly journals Nessun Dorma, a novel centralspindlin partner, is required for cytokinesis in Drosophila spermatocytes

2010 ◽  
Vol 191 (7) ◽  
pp. 1351-1365 ◽  
Author(s):  
Emilie Montembault ◽  
Wei Zhang ◽  
Marcin R. Przewloka ◽  
Vincent Archambault ◽  
Emeric W. Sevin ◽  
...  
Keyword(s):  
Essential Gene ◽  
Male Meiosis ◽  
Carbohydrate Binding ◽  
Ring Canal ◽  
Daughter Cells ◽  
High Affinity ◽  
Evolutionarily Conserved ◽  
Ring Canals ◽  
Female Germline

Cytokinesis, the final step of cell division, usually ends with the abscission of the two daughter cells. In some tissues, however, daughter cells never completely separate and remain interconnected by intercellular bridges or ring canals. In this paper, we report the identification and analysis of a novel ring canal component, Nessun Dorma (Nesd), isolated as an evolutionarily conserved partner of the centralspindlin complex, a key regulator of cytokinesis. Nesd contains a pectin lyase–like domain found in proteins that bind to polysaccharides, and we present evidence that it has high affinity for β-galactosides in vitro. Moreover, nesd is an essential gene in Drosophila melanogaster, in which it is required for completion of cytokinesis during male meiosis and possibly in female germline cells. Our findings indicate that Nesd is a novel carbohydrate-binding protein that functions together with centralspindlin in late cytokinesis, thus highlighting the importance of glycosylation in this process.

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