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.

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 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.

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 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.

Assembly of ring canals in the male germ line from structural components of the contractile ring

1996 ◽  
Vol 109 (12) ◽  
pp. 2779-2788 ◽  
Author(s):  
G.R. Hime ◽  
J.A. Brill ◽  
M.T. Fuller
Keyword(s):  
Germ Cells ◽  
Germ Line ◽  
Structural Components ◽  
Filamentous Actin ◽  
Ring Canal ◽  
Contractile Ring ◽  
Male Germ Line ◽  
Vesicular Structure ◽  
Ring Canals ◽  
Males And Females

Stable intercellular bridges called ring canals form following incomplete cytokinesis, and interconnect mitotically or meiotically related germ cells. We show that ring canals in Drosophila melanogaster males are surprisingly different from those previously described in females. Mature ring canal walls in males lack actin and appear to derive directly from structural proteins associated with the contractile ring. Ring canal assembly in males, as in females, initiates during cytokinesis with the appearance of a ring of phosphotyrosine epitopes at the site of the contractile ring. Following constriction, actin and myosin II disappear. However, at least four proteins present at the contractile ring remain: the three septins (Pnut, Sep1 and Sep2) and anillin. In sharp contrast, in ovarian ring canals, septins have not been detected, anillin is lost from mature ring canals and filamentous actin is a major component. In both males and females, a highly branched vesicular structure, termed the fusome, interconnects developing germ cells via the ring canals and is thought to coordinate mitotic germ cell divisions. We show that, in males, unlike females, the fusome persists and enlarges following cessation of the mitotic divisions, developing additional branches during meiosis. During differentiation, the fusome and its associated ring canals localize to the distal tip of the elongating spermatids.

Src64 is required for ovarian ring canal morphogenesis during Drosophila oogenesis

Development ◽  
1998 ◽  
Vol 125 (15) ◽  
pp. 2883-2892 ◽  
Author(s):  
G.S. Dodson ◽  
D.J. Guarnieri ◽  
M.A. Simon
Keyword(s):  
Tyrosine Phosphorylation ◽  
Cellular Proliferation ◽  
Src Family Kinases ◽  
Nurse Cells ◽  
Loss Of Function ◽  
Filamentous Actin ◽  
Ring Canal ◽  
Specific Product ◽  
Ring Canals ◽  
Egg Chambers

The Src family of protein tyrosine kinases have been implicated as important regulators of cellular proliferation, differentiation and function. In order to understand further the role of Src family kinases, we have generated loss-of-function mutations in Src64, one of two Src family kinases known in Drosophila melanogaster. Animals with reduced Src64 function develop normally and are fully viable. However, Src64 female flies have reduced fertility, which is associated with the incomplete transfer of cytoplasm from nurse cells to the developing oocyte. Analysis of Src64 egg chambers showed defects in the ring canals that interconnect the oocyte and its 15 associated nurse cells. Src64 ring canals fail to accumulate the high levels of tyrosine phosphorylation that are normally present. Despite the reduced tyrosine phosphorylation, known ring canal components such as filamentous actin, a ring canal-specific product of the hu-li tai shao gene, and the kelch protein localize properly. However, Src64 ring canals are reduced in size and frequently degenerate. These results indicate that Src64 is required for the proper growth and stability of the ovarian ring canals.

scholarly journals Drosophila Kelch Is an Oligomeric Ring Canal Actin Organizer

1997 ◽  
Vol 138 (4) ◽  
pp. 799-810 ◽  
Author(s):  
Douglas N. Robinson ◽  
Lynn Cooley
Keyword(s):  
Actin Filaments ◽  
Function Analysis ◽  
Cross Linking ◽  
Ring Canal ◽  
Amino Terminal ◽  
Btb Domain ◽  
Ring Canals ◽  
Repeat Domain ◽  
Additional Interaction ◽  
Necessary And Sufficient

Drosophila kelch has four protein domains, two of which are found in kelch-family proteins and in numerous nonkelch proteins. In Drosophila, kelch is required to maintain ring canal organization during oogenesis. We have performed a structure–function analysis to study the function of Drosophila kelch. The amino-terminal region (NTR) regulates the timing of kelch localization to the ring canals. Without the NTR, the protein localizes precociously and destabilizes the ring canals and the germ cell membranes, leading to dominant sterility. The amino half of the protein including the BTB domain mediates dimerization. Oligomerization through the amino half of kelch might allow cross-linking of ring canal actin filaments, organizing the inner rim cytoskeleton. The kelch repeat domain is necessary and sufficient for ring canal localization and likely mediates an additional interaction, possibly with actin.

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