Biology subjects

Quinlan, M. E.

Publications and source records attributed to Quinlan, M. E..

8 recordsLinked to original sources

bioRxiv2026

The Cappuccino interactome reveals an intracellular role for Semaphorin-2a in Drosophila oogenesis

The spatiotemporal regulation of an actin mesh during Drosophila oogenesis is essential for proper localization of cell polarity determinants that establish the future patterning of the embryo. Here, we reveal an unexpected role for Semaphorin-2a (Sema2a) in actin mesh regulation and oogenesis. Sema2a classically functions as a secreted guidance cue that binds its cognate Plexin-B (PlexB) receptor to establish neural circuits. In contrast, we find that Sema2a is expressed inside the germarium, germline, and follicle cells of the developing ovary. Sema2a mutants possess small ovaries that fail to develop past mid-oogenesis. We demonstrate that Sema2a interacts with Cappuccino (Capu), a key actin nucleator crucial for building the actin mesh in Drosophila oocytes. Sema2a inhibits the actin assembly activity of Capu in vitro. Furthermore, genetic interaction between Sema2a and Capu influences mesh density and disrupts oskar mRNA localization. PlexB mutants, however, exhibit wild-type size ovaries with oskar mRNA localization distinct from Sema2a mutants, confirming the non-canonical role of Sema2a in oogenesis. SummaryThis study reveals a novel interaction between the actin nucleator Cappuccino and the typically secreted neural guidance factor Semaphorin-2a. It is shown that Semaphorin-2a inhibits the actin polymerization activity of Cappuccino in vitro and play an intracellular role in oogenesis.

cell biology↗

bioRxiv2025

The impact of N-terminal dimerization on formin homology 1 (FH1) domain polymer dynamics and actin assembly

Many proteins contain intrinsically disordered regions (IDRs) that lack stable 3-dimensional structure. IDR behavior is poorly understood, leading to challenges for biochemical and computational analysis of IDR-containing proteins. Formins are a diverse set of homodimers containing an IDR -- the FH1 domain -- that facilitates polymerization of the cytoskeletal protein actin by increasing the local concentration of actin monomers at the actin assembly site. A commonly accepted model of formin-based actin polymerization involves a capture-and-deliver process: one or more binding sites (proline-rich motifs, PRMs) "capture" actin monomers and then "deliver" actin to the actin assembly site. There is evidence that formin FH1 domains are dimerized on both ends, but much research has been performed with formin constructs lacking the N-terminal dimerization site. Here, we ask: What happens when N-terminal dimerization is added to the standard model of formin-mediated actin assembly? We extend the kinetic model of FH1-mediated actin polymerization by incorporating a coarse-grain polymer model of FH1 domain dynamics, modeling the FH1 domain as a freely-jointed chain. We find that N-terminal dimerization can impact polymerization rates by modifying binding site accessibility and/or local concentration of binding sites (PRMs) at the actin assembly site (FH2 domain). Which effect dominates depends on kinetic parameters and formin properties such as FH1 domain length and binding site location. Additionally, we demonstrate that our model can be fit to experimental data and used to make predictions for the effects of N-terminal dimerization on a variety of formin family members. SignificanceIntrinsically disordered regions (IDRs) are common protein components that lack stable 3D structures and are thus difficult to study. Here, we develop a polymer-physics based computational model of the formin FH1 domain, an IDR involved in building the cells cytoskeleton. Some disease-associated mutations of formins occur in a region that is suspected to induce dimerization, forcing the FH1 domains to form a loop. Little is known about the loops prevalence, location, or impact on cytoskeletal assembly. Using simple polymer physics, we demonstrate that this dimerization can alter FH1 domain polymer dynamics and thus impact cytoskeletal assembly. These results not only highlight an important aspect of FH1-mediated cytoskeletal assembly, but also provide a framework for modeling IDRs.

biophysics↗

bioRxiv2024

Human formin FHOD3-mediated actin elongation is required for sarcomere integrity in cardiomyocytes

Contractility and cell motility depend on accurately controlled assembly of the actin cytoskeleton. Formins are a large group of actin assembly proteins that nucleate and elongate new actin filaments. Some formins may cap filaments while others sever or bundle filaments. The Formin HOmology Domain-containing protein (FHOD)-family of formins is critical to the formation of the fundamental contractile unit in muscle, the sarcomere. Specifically, mammalian FHOD3L plays an essential role in cardiomyocytes. Despite our knowledge of FHOD3Ls importance in cardiomyocytes, its biochemical and cellular activities remain poorly understood. It was proposed that FHOD-family formins act by capping and bundling, as opposed to assembling new filaments. Here, we demonstrate that FHOD3L nucleates actin and rapidly but briefly elongates filaments after temporarily pausing elongation. We designed function-separating mutants that enabled us to distinguish which biochemical roles are required in the cell. We found that human FHOD3Ls elongation activity, but not its nucleation, capping, or bundling activity, is necessary for proper sarcomere formation and contractile function in neonatal rat ventricular myocytes. The results of this work provide new insight into the mechanisms by which formins build specific structures and will contribute to knowledge regarding how cardiomyopathies arise from defects in sarcomere formation and maintenance.

cell biology↗

bioRxiv2024

Localization of Drosophila formin, Cappuccino, influences posterior oocyte organization

AbstractCappuccino (Capu) and Spire build actin networks in numerous systems, including the mouse oocyte, melanocytes, and the Drosophila oocyte. As observed in mammalian systems, the localization of the Capu homologues (FMN1/2), influences the function of the actin network. Therefore, we established and interrogated the impact of altering Capus localization in the Drosophila oocyte to better understand its role and that of the actin mesh it builds. This mesh restricts bulk cytoplasmic flows, streaming, but otherwise remains undescribed functionally. Using a gene specific driver, capu-Gal4, to better study Capu transgenes, we found that fertility was markedly decreased when restricting Capu to membranes in the oocyte, although its canonical role in actin mesh assembly was apparently unaltered. Instead, we observed a defect in posterior anchoring of the mRNA oskar during mid-oogenesis. However, the defect did not fall into the traditional posterior group phenotype. The data suggest that Capu, independently of Spire, tethers the posterior determinants to the cortex but does not anchor them to each other, supporting that Capu localization influences the posterior oocyte organization.

developmental biology↗

bioRxiv2023

Formin tails act as a switch, inhibiting or enhancing processive actin elongation

Formins are large, multidomain proteins that nucleate new actin filaments and accelerate elongation through a processive interaction with the barbed end of filaments. Their actin assembly activity is generally attributed to their eponymous Formin Homology (FH) 1 and 2 domains; however, evidence is mounting that regions outside of the FH1FH2 stretch also tune actin assembly. Here, we explore the underlying contributions of the tail domain, which spans the sequence between the FH2 domain and the C-terminus of formins. Tails vary in length from [~]0 to >200 residues and contain a number of recognizable motifs. The most well-studied motif is the [~]15 residue long diaphanous autoregulatory domain (DAD). The DAD mediates all or nothing regulation of actin assembly through an intramolecular interaction with the diaphanous inhibitory domain (DID) in the N-terminal half of the protein. Multiple reports demonstrate that the tail can enhance both nucleation and processivity. In this study, we provide a high-resolution view of the alternative splicing encompassing the tail in the Formin Homology Domain-(Fhod) family of formins during development. While four distinct tails are predicted, we found significant levels of only two of these. We characterized the biochemical effects of the different tails. Surprisingly, the two highly expressed Fhod-tails inhibit processive elongation and diminish the nucleation and elongation rates, while a third supports activity. These findings demonstrate a new mechanism of modulating actin assembly by formins and support the model that splice variants are specialized to build distinct actin structures during development.

biochemistry↗

bioRxiv2019

Spire stimulates nucleation by Cappuccino and binds both ends of actin filaments

An actin mesh fills both mouse and fly oocytes. The meshes are built by a conserved mechanism and used to establish polarity. Two actin nucleators, Spire and Cappuccino, collaborate to build actin filaments that connect vesicles and the cortex. Direct interaction between Spire and Cappuccino is required for in vitro synergistic actin assembly; however, we understand little about why the interaction is necessary. To mimic the geometry of Spire and Cappuccino in vivo, we immobilized Spire on beads. We found that increased nucleation is a major part of synergy and that Spire alone binds both barbed- and pointed-ends of actin filaments. We identified Spires barbed-end binding domain. Partial rescue of fertility by a loss-of-function mutant indicates that barbed-end binding is not necessary for Spires in vivo function, but that it may play a role under normal circumstances. We propose that Spire stimulates nucleation by Cappuccino in a manner similar to the collaboration between APC and mDia1.\n\nSummaryActin nucleators Cappuccino and Spire collaborate to build an actin mesh in oocytes. Data demonstrate that the collaboration leads to synergistic actin nucleation, as opposed to elongation. Further, Spire binds both ends of polar, actin filaments, resolving a long-outstanding question.

biochemistry↗

bioRxiv2017

The Drosophila Formin Fhod Nucleates Actin Filaments

Formins are a conserved group of proteins that nucleate and processively elongate actin filaments. Among them, the formin homology domain-containing protein (FHOD) family of formins contributes to contractility of striated muscle and cell motility in several contexts. However, the mechanisms by which they carry out these functions remain poorly understood. Unlike other formins, mammalian FHOD1 and FHOD3 do not accelerate actin assembly in vitro, and have instead been suggested to act as barbed end cappers or bundlers. Here, we show that purified Drosophila Fhod, in contrast with the mammalian homologues, potently accelerates actin assembly by nucleation. We found that Fhod binds tightly to barbed ends, where it slows elongation in the absence of profilin and allows elongation in the presence of profilin. Fhod protects barbed ends from capping protein, but dissociates from barbed ends relatively quickly. Finally, we used cosedimentation assays to determine that Fhod binds the sides of actin filaments and bundles filaments. This work establishes that Fhod shares the capacity of other formins to nucleate and bundle actin filaments, but is notably less effective at processively elongating barbed ends.

cell biology↗

bioRxiv2017

The Neuron Specific Formin Delphilin Nucleates Actin Filaments but Does Not Enhance Elongation

The formin Delphilin binds the glutamate receptor, GluR{delta}2, in dendritic spines of Purkinje cells. Both proteins play a role in learning. To understand how Delphilin functions in neurons, we studied the actin assembly properties of this formin. Formins have a conserved formin homology 2 domain, which nucleates and remains associated with the fast growing end of actin filaments, influencing filament growth with input from the adjacent formin homology 1 domain. The strength of nucleation and elongation varies widely across formins. Additionally, most formins have conserved domains that regulate actin assembly through an intramolecular interaction. Delphilin is distinct from other formins in several ways: its expression is limited to Purkinje cells; it lacks autoinhibitory domains; its formin homology 1 domains has minimal proline-rich sequence. We found that Delphilin is an actin nucleator that does not accelerate elongation, although it binds tightly to the barbed end of filaments. In addition, Delphilin exhibits a preference for actin isoforms, which has not been described or systematically studied in other formins. Finally, Delphilin is the first formin studied that is not regulated by intramolecular interactions. We speculate how the activity we observe is consistent with its localization in the small dendritic spines.\n\nAbbreviations: FH1, formin homology 1; FH2, formin homology 2; DID, diaphanous inhibitory domain; DAD, diaphanous autoinhibitory domain; LTD, long term depression; PSD, post synaptic density; PDZ, post synaptic density protein (PSD95), Drosophila disc large tumor suppressor (Dlg1), and zonula occludens-1 protein (zo-1).

biochemistry↗