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

Andrew M. Hudson; Katelynn M. Mannix; Julianne A. Gerdes; Molly C. Kottemann; Lynn Cooley

doi:10.1242/dev.169219

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

10.1101/2020.04.24.060186 ◽

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.

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

10.1101/315077 ◽

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.

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

Genetics ◽

10.1534/genetics.120.303629 ◽

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.

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Filamin Is Required for Ring Canal Assembly and Actin Organization during Drosophila Oogenesis

The Journal of Cell Biology ◽

10.1083/jcb.146.5.1061 ◽

1999 ◽

Vol 146 (5) ◽

pp. 1061-1074 ◽

Cited By ~ 58

Author(s):

Min-gang Li ◽

Madeline Serr ◽

Kevin Edwards ◽

Susan Ludmann ◽

Daisuke Yamamoto ◽

...

Keyword(s):

Cell Migration ◽

Actin Cytoskeleton ◽

Binding Proteins ◽

Genetic Model ◽

Membrane Integrity ◽

Actin Binding ◽

Female Sterility ◽

Ring Canal ◽

Actin Binding Proteins

The remodeling of the actin cytoskeleton is essential for cell migration, cell division, and cell morphogenesis. Actin-binding proteins play a pivotal role in reorganizing the actin cytoskeleton in response to signals exchanged between cells. In consequence, actin-binding proteins are increasingly a focus of investigations into effectors of cell signaling and the coordination of cellular behaviors within developmental processes. One of the first actin-binding proteins identified was filamin, or actin-binding protein 280 (ABP280). Filamin is required for cell migration (Cunningham et al. 1992), and mutations in human α-filamin (FLN1; Fox et al. 1998) are responsible for impaired migration of cerebral neurons and give rise to periventricular heterotopia, a disorder that leads to epilepsy and vascular disorders, as well as embryonic lethality. We report the identification and characterization of a mutation in Drosophila filamin, the homologue of human α-filamin. During oogenesis, filamin is concentrated in the ring canal structures that fortify arrested cleavage furrows and establish cytoplasmic bridges between cells of the germline. The major structural features common to other filamins are conserved in Drosophila filamin. Mutations in Drosophila filamin disrupt actin filament organization and compromise membrane integrity during oocyte development, resulting in female sterility. The genetic and molecular characterization of Drosophila filamin provides the first genetic model system for the analysis of filamin function and regulation during development.

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A role for the actin cytoskeleton in the hormonal and growth-factor-mediated activation of protein kinase B

Biochemical Journal ◽

10.1042/0264-6021:3530735v ◽

2001 ◽

Vol 353 (3) ◽

pp. 735

Author(s):

K. PEYROLLIER ◽

E. HAJDUCH ◽

A. GRAY ◽

G. J. LITHERLAND ◽

A. R. PRESCOTT ◽

...

Keyword(s):

Growth Factor ◽

Protein Kinase ◽

Actin Cytoskeleton ◽

Protein Kinase B

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Spatial control of actin-based motility through plasmalemmal PtdIns(4,5)P2-rich raft assemblies.

Biochemical Society Symposium ◽

10.1042/bss0720119 ◽

2005 ◽

Vol 72 ◽

pp. 119-127 ◽

Cited By ~ 19

Author(s):

Tamara Golub ◽

Caroni Pico

Keyword(s):

Protein Kinase ◽

Cell Surface ◽

Actin Cytoskeleton ◽

Lipid Rafts ◽

Patch Clustering ◽

Pkc Protein Kinase C ◽

Membrane Bound ◽

And Function ◽

Cytoskeleton Dynamics ◽

Syndrome Protein

The interactions of cells with their environment involve regulated actin-based motility at defined positions along the cell surface. Sphingolipid- and cholesterol-dependent microdomains (rafts) order proteins at biological membranes, and have been implicated in most signalling processes at the cell surface. Many membrane-bound components that regulate actin cytoskeleton dynamics and cell-surface motility associate with PtdIns(4,5)P2-rich lipid rafts. Although raft integrity is not required for substrate-directed cell spreading, or to initiate signalling for motility, it is a prerequisite for sustained and organized motility. Plasmalemmal rafts redistribute rapidly in response to signals, triggering motility. This process involves the removal of rafts from sites that are not interacting with the substrate, apparently through endocytosis, and a local accumulation at sites of integrin-mediated substrate interactions. PtdIns(4,5)P2-rich lipid rafts can assemble into patches in a process depending on PtdIns(4,5)P2, Cdc42 (cell-division control 42), N-WASP (neural Wiskott-Aldrich syndrome protein) and actin cytoskeleton dynamics. The raft patches are sites of signal-induced actin assembly, and their accumulation locally promotes sustained motility. The patches capture microtubules, which promote patch clustering through PKA (protein kinase A), to steer motility. Raft accumulation at the cell surface, and its coupling to motility are influenced greatly by the expression of intrinsic raft-associated components that associate with the cytosolic leaflet of lipid rafts. Among them, GAP43 (growth-associated protein 43)-like proteins interact with PtdIns(4,5)P2 in a Ca2+/calmodulin and PKC (protein kinase C)-regulated manner, and function as intrinsic determinants of motility and anatomical plasticity. Plasmalemmal PtdIns(4,5)P2-rich raft assemblies thus provide powerful organizational principles for tight spatial and temporal control of signalling in motility.

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