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.

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 The Arp2/3 complex and the formin, Diaphanous, are both required to regulate the size of germline ring canals in the developing egg chamber

2019 ◽  
Author(s):  
Josephine Thestrup ◽  
Marina Tipold ◽  
Alexandra Kindred ◽  
Kara Stark ◽  
Travis Curry ◽  
...  
Keyword(s):  
Fruit Fly ◽  
Actin Binding ◽  
Nurse Cells ◽  
Intercellular Bridge ◽  
Ring Canal ◽  
Contractile Ring ◽  
Intercellular Bridges ◽  
Large Size ◽  
Ring Canals ◽  
Egg Chamber

AbstractIntercellular bridges are an essential structural feature found in both germline and somatic cells throughout the animal kingdom. Because of their large size, the germline intercellular bridges, or ring canals, in the developing fruit fly egg chamber are an excellent model to study the formation, stabilization, and expansion of these structures. Within the egg chamber, the germline ring canals connect 15 supporting nurse cells to the developing oocyte, facilitating the transfer of materials required for successful oogenesis. The ring canals are derived from a stalled actomyosin contractile ring; once formed, additional actin and actin-binding proteins are recruited to the ring to support the 20-fold expansion that accompanies oogenesis. These behaviors provide a unique model system to study the actin regulators that control incomplete cytokinesis, intercellular bridge formation, and expansion. By temporally controlling their expression in the germline, we have demonstrated that the Arp2/3 complex and the formin, Diaphanous (Dia), coordinately regulate ring canal size and expansion throughout oogenesis. Dia is required for successful incomplete cytokinesis and the initial stabilization of the germline ring canals. Once the ring canals have formed, the Arp2/3 complex and Dia cooperate to determine ring canal size and maintain their stability. Our data suggest that the nurse cells must maintain a precise balance between the activity of these two nucleators during oogenesis.

Drosophila quail, a villin-related protein, bundles actin filaments in apoptotic nurse cells

Development ◽  
1999 ◽  
Vol 126 (24) ◽  
pp. 5645-5657 ◽  
Author(s):  
N. Matova ◽  
S. Mahajan-Miklos ◽  
M.S. Mooseker ◽  
L. Cooley
Keyword(s):  
Actin Filaments ◽  
Nurse Cell ◽  
Calcium Concentration ◽  
Developmental Model ◽  
Free Calcium ◽  
Nurse Cells ◽  
Filamentous Actin ◽  
Actin Bundle ◽  
High Calcium ◽  
Egg Chambers

Drosophila Quail protein is required for the completion of fast cytoplasm transport from nurse cells to the oocyte, an event critical for the production of viable oocytes. The abundant network of cytoplasmic filamentous actin, established at the onset of fast transport, is absent in quail mutant egg chambers. Previously, we showed that Quail is a germline-specific protein with sequence homology to villin, a vertebrate actin-regulating protein. In this study, we combined biochemical experiments with observations in egg chambers to define more precisely the function of this protein in the regulation of actin-bundle assembly in nurse cells. We report that recombinant Quail can bind and bundle filamentous actin in vitro in a manner similar to villin at a physiological calcium concentration. In contrast to villin, Quail is unable to sever or cap filamentous actin, or to promote nucleation of new actin filaments at a high calcium concentration. Instead, Quail bundles the filaments regardless of the calcium concentration. In vivo, the assembly of nurse-cell actin bundles is accompanied by extensive perforation of the nurse-cell nuclear envelopes, and both of these phenomena are manifestations of nurse-cell apoptosis. To investigate whether free calcium levels are affected during apoptosis, we loaded egg chambers with the calcium indicator Indo-1. Our observations indicate a rise in free calcium in the nurse-cell cytoplasm coincident with the permeabilization of the nuclear envelopes. We also show that human villin expressed in the Drosophila germline could sense elevated cytoplasmic calcium; in nurse cells with reduced levels of Quail protein, villin interfered with actin-bundle stability. We conclude that Quail efficiently assembles actin filaments into bundles in nurse cells and maintains their stability under fluctuating free calcium levels. We also propose a developmental model for the fast phase of cytoplasm transport incorporating findings presented in this study.

scholarly journals Formation of actin filament bundles in the ring canals of developing Drosophila follicles.

1996 ◽  
Vol 133 (1) ◽  
pp. 61-74 ◽  
Author(s):  
L G Tilney ◽  
M S Tilney ◽  
G M Guild
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Actin Binding ◽  
Nurse Cells ◽  
Ring Canal ◽  
Contractile Ring ◽  
Thin Sections ◽  
Development Stages ◽  
Ring Canals ◽  
Filament Bundles

Growing the intracellular bridges that connect nurse cells with each o ther and to the developing oocyte is vital for egg development. These ring canals increase from 0.5 microns in diameter at stage 2 to 10 microns in diameter at stage 11. Thin sections cut horizontally as you would cut a bagel, show that there is a layer of circumferentially oriented actin filaments attached to the plasma membrane at the periphery of each canal. By decoration with subfragment 1 of myosin we find actin filaments of mixed polarities in the ring such as found in the "contractile ring" formed during cytokinesis. In vertical sections through the canal the actin filaments appear as dense dots. At stage 2 there are 82 actin filaments in the ring, by stage 6 there are 717 and by stage 10 there are 726. Taking into account the diameter, this indicates that there is 170 microns of actin filaments/canal at stage 2 (pi x 0.5 microns x 82), 14,000 microns at stage 9 and approximately 23,000 microns at stage 11 or one inch of actin filament! The density of actin filaments remains unchanged throughout development. What is particularly striking is that by stages 4-5, the ring of actin filaments has achieved its maximum thickness, even though the diameter has not yet increased significantly. Thereafter, the diameter increases. Throughout development, stages 2-11, the canal length also increases. Although the density (number of actin filaments/micron2) through a canal remains constant from stage 5 on, the actin filaments appear as a net of interconnected bundles. Further information on this net of bundles comes from studying mutant animals that lack kelch, a protein located in the ring canal that has homology to the actin binding protein, scruin. In this mutant, the actin filaments form normally but individual bundles that comprise the fibers of the net are not bound tightly together. Some bundles enter into the ring canal lumen but do not completely occlude the lumen. all these observations lay the groundwork for our understanding of how a noncontractile ring increases in thickness, diameter, and length during development.

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.

An A-kinase anchoring protein is required for Protein kinase A regulatory subunit localization and morphology of actin structures during oogenesis inDrosophila

Development ◽  
2002 ◽  
Vol 129 (19) ◽  
pp. 4423-4433 ◽  
Author(s):  
Stephen M. Jackson ◽  
Celeste A. Berg
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Membrane Integrity ◽  
Regulatory Subunit ◽  
Small Ring ◽  
Nurse Cells ◽  
Ring Canal ◽  
Ring Canals ◽  
Germline Cells ◽  
Kinase A

Protein kinase A (PKA) holoenzyme is anchored to specific subcellular regions by interactions between regulatory subunits (Pka-R) and A-kinase anchoring proteins (AKAPs). We examine the functional importance of PKA anchoring during Drosophila oogenesis by analyzing membrane integrity and actin structures in mutants with disruptions in Akap200, an AKAP. In wild-type ovaries, Pka-RII and Akap200 localized to membranes and to the outer rim of ring canals, actin-rich structures that connect germline cells. In Akap200 mutant ovaries, Pka-RII membrane localization decreased, leading to a destabilization of membrane structures and the formation of binucleate nurse cells. Defects in membrane integrity could be mimicked by expressing a constitutively active PKA catalytic subunit (Pka-C) throughout germline cells. Unexpectedly, nurse cells in Akap200 mutant ovaries also had enlarged, thin ring canals. In contrast, overexpressing Akap200 in the germline resulted in thicker, smaller ring canals. To investigate the role of Akap200 in regulating ring canal growth, we examined genetic interactions with other genes that are known to regulate ring canal morphology. Akap200 mutations suppressed the small ring canal phenotype produced by Src64B mutants, linking Akap200 with the non-receptor tyrosine kinase pathway. Together, these results provide the first evidence that PKA localization is required for morphogenesis of actin structures in an intact organism.

scholarly journals Inhibition of Src Family Kinases by a Combinatorial Action of 5′-AMP and Small Heat Shock Proteins, Identified from the Adult Heart

1999 ◽  
Vol 19 (10) ◽  
pp. 6858-6871 ◽  
Author(s):  
Vijaykumar S. Kasi ◽  
Dhandapani Kuppuswamy
Keyword(s):  
Heat Shock ◽  
Tyrosine Phosphorylation ◽  
Cellular Proliferation ◽  
Catalytic Domain ◽  
Direct Interaction ◽  
Small Heat Shock Protein ◽  
Peptide Sequencing ◽  
Src Family Kinases

ABSTRACT Src family kinases are implicated in cellular proliferation and transformation. Terminally differentiated myocytes have lost the ability to proliferate, indicating the existence of a down-regulatory mechanism(s) for these mitogenic kinases. Here we show that feline cardiomyocyte lysate contains thermostable components that inhibit c-Src kinase in vitro. This inhibitory activity, present predominantly in heart tissue, involves two components acting combinatorially. After purification by sequential chromatography, one component was identified by mass and nuclear magnetic resonance spectroscopies as 5′-AMP, while the other was identified by peptide sequencing as a small heat shock protein (sHSP). 5′-AMP and to a lesser extent 5′-ADP inhibit c-Src when combined with either HSP-27 or HSP-32. Other HSPs, including αB-crystallin, HSP-70, and HSP-90, did not exhibit this effect. The inhibition, observed preferentially on Src family kinases and independent of the Src tyrosine phosphorylation state, occurs via a direct interaction of the c-Src catalytic domain with the inhibitory components. Our study indicates that sHSPs increase the affinity of 5′-AMP for the c-Src ATP binding site, thereby facilitating the inhibition. In vivo, elevation of ATP levels in the cardiomyocytes results in the tyrosine phosphorylation of cellular proteins including c-Src at the activatory site, and this effect is blocked when the 5′-AMP concentration is raised. Thus, this study reveals a novel role for sHSPs and 5′-AMP in the regulation of Src family kinases, presumably for the maintenance of the terminally differentiated state.

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 INTERCELLULAR MIGRATION OF CENTRIOLES IN THE GERMARIUM OF DROSOPHILA MELANOGASTER

1970 ◽  
Vol 45 (2) ◽  
pp. 306-320 ◽  
Author(s):  
Anthony P. Mahowald ◽  
Joan M. Strassheim
Keyword(s):  
Follicle Cell ◽  
Cell Cluster ◽  
Follicle Cells ◽  
Oocyte Nucleus ◽  
Nurse Cells ◽  
Serial Sections ◽  
Cell Border ◽  
Ring Canals ◽  
Egg Chambers ◽  
Stage 1

A cluster of centrioles has been found in the early Drosophila oocyte. Since the oocyte is connected to 15 nurse cells by a system of intercellular bridges or ring canals, the possibility that the cluster of centrioles arose in the germarium from an intercellular migration of centrioles from the nurse cells to the oocyte was analyzed in serial sections for the electron microscope. Initially, all of the 16 cells of the future egg chambers possess centrioles, which are located in a juxtanuclear position. At the time the 16 cell cluster becomes arranged in a lens-shaped layer laterally across the germarium, the centrioles lose their juxtanuclear position and move towards the oocyte. By the time the 16 cell cluster of cells is surrounded by follicle cells (Stage 1), between 14 and 17 centrioles are found in the oocyte. Later, these centrioles become located between the oocyte nucleus and the follicle cell border and become aggregated into a cluster less than 1.5 µ in its largest dimension. The fate of these centrioles in the oocyte is not known. The fine structure of the germarium and the early oocyte is also described.

scholarly journals Precise levels of the Drosophila adaptor protein Dreadlocks maintain the size and stability of germline ring canals

2021 ◽  
Vol 134 (8) ◽  
Author(s):  
Kara Stark ◽  
Olivia Crowe ◽  
Lindsay Lewellyn
Keyword(s):  
Genetic Interaction ◽  
Adaptor Protein ◽  
Fold Increase ◽  
Fruit Fly ◽  
Sh2 Domain ◽  
Nurse Cells ◽  
Ring Canal ◽  
Intercellular Bridges ◽  
Canal Diameter ◽  
Ring Canals

ABSTRACT Intercellular bridges are essential for fertility in many organisms. The developing fruit fly egg has become the premier model system to study intercellular bridges. During oogenesis, the oocyte is connected to supporting nurse cells by relatively large intercellular bridges, or ring canals. Once formed, the ring canals undergo a 20-fold increase in diameter to support the movement of materials from the nurse cells to the oocyte. Here, we demonstrate a novel role for the conserved SH2/SH3 adaptor protein Dreadlocks (Dock) in regulating ring canal size and structural stability in the germline. Dock localizes at germline ring canals throughout oogenesis. Loss of Dock leads to a significant reduction in ring canal diameter, and overexpression of Dock causes dramatic defects in ring canal structure and nurse cell multinucleation. The SH2 domain of Dock is required for ring canal localization downstream of Src64 (also known as Src64B), and the function of one or more of the SH3 domains is necessary for the strong overexpression phenotype. Genetic interaction and localization studies suggest that Dock promotes WASp-mediated Arp2/3 activation in order to determine ring canal size and regulate growth. This article has an associated First Person interview with the first author of the paper.

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