Prostaglandins temporally regulate cytoplasmic actin bundle formation during Drosophila oogenesis

Prostaglandins (PGs)—lipid signals produced downstream of cyclooxygenase (COX) enzymes—regulate actin dynamics in cell culture and platelets, but their roles during development are largely unknown. Here we define a new role for Pxt, the Drosophila COX-like enzyme, in regulating the actin cytoskeleton—temporal restriction of actin remodeling during oogenesis. PGs are required for actin filament bundle formation during stage 10B (S10B). In addition, loss of Pxt results in extensive early actin remodeling, including actin filaments and aggregates, within the posterior nurse cells of S9 follicles; wild-type follicles exhibit similar structures at a low frequency. Hu li tai shao (Hts-RC) and Villin (Quail), an actin bundler, localize to all early actin structures, whereas Enabled (Ena), an actin elongation factor, preferentially localizes to those in pxt mutants. Reduced Ena levels strongly suppress early actin remodeling in pxt mutants. Furthermore, loss of Pxt results in reduced Ena localization to the sites of bundle formation during S10B. Together these data lead to a model in which PGs temporally regulate actin remodeling during Drosophila oogenesis by controlling Ena localization/activity, such that in S9, PG signaling inhibits, whereas at S10B, it promotes Ena-dependent actin remodeling.     

The pros and cons of common actin labeling tools for visualizing actin dynamics during Drosophila oogenesis

Dynamic remodeling of the actin cytoskeleton is required for both development and tissue homeostasis. While fixed image analysis has provided significant insight into such events, a complete understanding of cytoskeletal dynamics requires live imaging. Numerous tools for the live imaging of actin have been generated by fusing the actin-binding domain from an actin-interacting protein to a fluorescent protein. Here we comparatively assess the utility of three such tools – Utrophin, Lifeact, and F-tractin – for characterizing the actin remodeling events occurring within the germline-derived nurse cells during Drosophila mid-oogenesis or follicle development. Specifically, we used the UAS/GAL4 system to express these tools at different levels and in different cells, and analyzed these tools for effects on fertility, alterations in the actin cytoskeleton, and ability to label filamentous actin (F-actin) structures by both fixed and live imaging. While both Utrophin and Lifeact robustly label F-actin structures within the Drosophila germline, when strongly expressed they cause sterility and severe actin defects including cortical actin breakdown resulting in multi-nucleate nurse cells, early F-actin filament and aggregate formation during stage 9 (S9), and disorganized parallel actin filament bundles during stage 10B (S10B). However, by using a weaker germline GAL4 driver in combination with a higher temperature, Utrophin can label F-actin with minimal defects. Additionally, strong Utrophin expression within the germline causes F-actin formation in the nurse cell nuclei and germinal vesicle during mid-oogenesis. Similarly, Lifeact expression results in nuclear F-actin only within the germinal vesicle. F-tractin expresses at a lower level than the other two labeling tools, but labels cytoplasmic F-actin structures well without causing sterility or striking actin defects. Together these studies reveal how critical it is to evaluate the utility of each actin labeling tool within the tissue and cell type of interest in order to identify the tool that represents the best compromise between acceptable labeling and minimal disruption of the phenomenon being observed. In this case, we find that F-tractin, and perhaps Utrophin, when Utrophin expression levels are optimized to label efficiently without causing actin defects, can be used to study F-actin dynamics within the Drosophila nurse cells.     

Prostaglandins regulate nuclear localization of Fascin and its function in nucleolar architecture

Fascin, a highly conserved actin-bundling protein, localizes and functions at new cellular sites in both Drosophila and multiple mammalian cell types. During Drosophila follicle development, in addition to being cytoplasmic, Fascin is in the nuclei of the germline-derived nurse cells during stages 10B–12 (S10B–12) and at the nuclear periphery during stage 13 (S13). This localization is specific to Fascin, as other actin-binding proteins, Villin and Profilin, do not exhibit the same subcellular distribution. In addition, localization of fascin1 to the nucleus and nuclear periphery is observed in multiple mammalian cell types. Thus the regulation and function of Fascin at these new cellular locations is likely to be highly conserved. In Drosophila, loss of prostaglandin signaling causes a global reduction in nuclear Fascin and a failure to relocalize to the nuclear periphery. Alterations in nuclear Fascin levels result in defects in nucleolar morphology in both Drosophila follicles and cultured mammalian cells, suggesting that nuclear Fascin plays an important role in nucleolar architecture. Given the numerous roles of Fascin in development and disease, including cancer, our novel finding that Fascin has functions within the nucleus sheds new light on the potential roles of Fascin in these contexts.     

Fascin: A key regulator of cytoskeletal dynamics

Fascin is a 55 kDa actin-bundling protein and is an important regulatory element in the maintenance and stability of parallel bundles of filamentous actin in a variety of cellular contexts. Regulation of fascin function is under the control of a number of different signalling pathways that act in concert to spatially regulate the actin-binding properties of this protein. The ability of fascin to bind and bundle actin plays a central role in the regulation of cell adhesion, migration and invasion. Fascin has received considerable attention recently as an emerging key prognostic marker of metastatic disease. Studies are now underway to better understand the precise regulation of this protein in the context of tumour progression and to investigate fascin as a potential therapeutic target for a number of forms of cancer.     

Enabled and Capping protein play important roles in shaping cell behavior during Drosophila oogenesis

During development, cells craft an impressive array of actin-based structures, mediating events as diverse as cytokinesis, apical constriction, and cell migration. One challenge is to determine how cells regulate actin assembly and disassembly to carry out these cell behaviors. During Drosophila oogenesis diverse cell behaviors are seen in the soma and germline. We used oogenesis to explore developmental roles of two important actin regulators: Enabled/VASP proteins and Capping protein. We found that Enabled plays an important role in cortical integrity of nurse cells, formation of robust bundled actin filaments in late nurse cells that facilitate nurse cell dumping, and migration of somatic border cells. During nurse cell dumping, Enabled localizes to barbed ends of the nurse cell actin filaments, suggesting its mechanism of action. We further pursued this mechanism using mutant Enabled proteins, each affecting one of its protein domains. These data suggest critical roles for the EVH2 domain and its tetramerization subdomain, while the EVH1 domain appears less critical. Enabled appears to be negatively regulated during oogenesis by Abelson kinase. We also explored the function of Capping protein. This revealed important roles in oocyte determination, nurse cell cortical integrity and nurse cell dumping, and support the idea that Capping protein and Enabled act antagonistically during dumping. Together these data reveal places that these actin regulators shape oogenesis.     

Rho1 regulates adherens junction remodeling by promoting recycling endosome formation through activation of myosin II

Once adherens junctions (AJs) are formed between polarized epithelial cells they must be maintained because AJs are constantly remodeled in dynamic epithelia. AJ maintenance involves endocytosis and subsequent recycling of E-cadherin to a precise location along the basolateral membrane. In the Drosophila pupal eye epithelium, Rho1 GTPase regulates AJ remodeling through Drosophila E-cadherin (DE-cadherin) endocytosis by limiting Cdc42/Par6/aPKC complex activity. We demonstrate that Rho1 also influences AJ remodeling by regulating the formation of DE-cadherin–containing, Rab11-positive recycling endosomes in Drosophila postmitotic pupal eye epithelia. This effect of Rho1 is mediated through Rok-dependent, but not MLCK-dependent, stimulation of myosin II activity yet independent of its effects upon actin remodeling. Both Rho1 and pMLC localize on endosomal vesicles, suggesting that Rho1 might regulate the formation of recycling endosomes through localized myosin II activation. This work identifies spatially distinct functions for Rho1 in the regulation of DE-cadherin–containing vesicular trafficking during AJ remodeling in live epithelia.     

Roles of Peroxinectin in PGE2-Mediated Cellular Immunity in Spodoptera exigua

Background

Prostaglandins (PGs) mediate insect immune responses to infections and invasions. Although the presence of PGs has been confirmed in several insect species, their biosynthesis in insects remains a conundrum because orthologs of the mammalian cyclooxygenases (COXs) have not been found in the known insect genomes. PG-mediated immune reactions have been documented in the beet armyworm, Spodoptera exigua. The purpose of this research is to identify the source of PGs in S. exigua.

Principal Findings

Peroxidases (POXs) are a sister group of COX genes. Ten putative POXs (SePOX-A ∼ SePOX-J) were expressed in S. exigua. Expressions of SePOX-F and -H were induced by bacterial challenge and expressed in the hemocytes and the fat body. RNAi of each POX was performed by hemocoelic injection of their specific double-stranded RNAs. dsPOX-F or, separately, dsPOX-H, but not the other eight dsRNA constructs, specifically suppressed hemocyte-spreading behavior and nodule formation; these two reactions were also inhibited by aspirin, a COX inhibitor. PGE₂, but not arachidonic acid, treatment rescued the immunosuppression. Sequence analysis indicated that both POX genes were clustered with peroxinectin (Pxt) and their cognate proteins shared some conserved domains corresponding to the Pxt of Drosophila melanogaster.

Conclusions

SePOX-F and -H are Pxt-like genes associated with PG biosynthesis in S. exigua.     

Actin Filament Cables in Drosophila Nurse Cells Are Composed of Modules That Slide Passively Past One Another during Dumping

At a late stage in Drosophila oogenesis, nurse cells rapidly expel their cytoplasm into the oocyte via intracellular bridges by a process called nurse cell dumping. Before dumping, numerous cables composed of actin filaments appear in the cytoplasm and extend inward from the plasma membrane toward the nucleus. This actin cage prevents the nucleus, which becomes highly lobed, from physically blocking the intracellular bridges during dumping. Each cable is composed of a linear series of modules composed of ~25 cross-linked actin filaments. Adjacent modules overlap in the cable like the units of an extension ladder. During cable formation, individual modules are nucleated from the cell surface as microvilli, released, and then cross-linked to an adjacent forming module. The filaments in all the modules in a cable are unidirectionally polarized. During dumping as the volume of the cytoplasm decreases, the nucleus to plasma membrane distance decreases, compressing the actin cables that shorten as adjacent modules slide passively past one another just as the elements of an extension ladder slide past one another for storage. In Drosophila, the modular construction of actin cytoskeletons seems to be a generalized strategy. The behavior of modular actin cytoskeletons has implications for other actin-based cytoskeletal systems, e.g., those involved in Listeria movement, in cell spreading, and in retrograde flow in growth cones and fibroblasts.     

The actin regulators Enabled and Diaphanous direct distinct protrusive behaviors in different tissues during Drosophila development

Actin-based protrusions are important for signaling and migration during development and homeostasis. Defining how different tissues in vivo craft diverse protrusive behaviors using the same genomic toolkit of actin regulators is a current challenge. The actin elongation factors Diaphanous and Enabled both promote barbed-end actin polymerization and can stimulate filopodia in cultured cells. However, redundancy in mammals and Diaphanous’ role in cytokinesis limited analysis of whether and how they regulate protrusions during development. We used two tissues driving Drosophila dorsal closure—migratory leading-edge (LE) and nonmigratory amnioserosal (AS) cells—as models to define how cells shape distinct protrusions during morphogenesis. We found that nonmigratory AS cells produce filopodia that are morphologically and dynamically distinct from those of LE cells. We hypothesized that differing Enabled and/or Diaphanous activity drives these differences. Combining gain- and loss-of-function with quantitative approaches revealed that Diaphanous and Enabled each regulate filopodial behavior in vivo and defined a quantitative “fingerprint”—the protrusive profile—which our data suggest is characteristic of each actin regulator. Our data suggest that LE protrusiveness is primarily Enabled driven, whereas Diaphanous plays the primary role in the AS, and reveal each has roles in dorsal closure, but its robustness ensures timely completion in their absence.     

Fascin,may the Forked be with you

The FGFR pathway triggers a wide range of key biological responses. Among others, the Breathless (Btl, Drosophila FGFR1) receptor cascade promotes cell migration during embryonic tracheal system development. However, how the actin cytoskeleton responds to Btl pathway activation to induce cell migration has remained largely unclear. Our recent results shed light into this issue by unveiling a link between the actin-bundling protein Singed (Sn) and the Btl pathway. We showed that the Btl pathway regulates sn, which leads to the stabilization of the actin bundles required for filopodia formation and actin cytoskeleton rearrangement. This regulation contributes to tracheal migration, tracheal branch fusion and tracheal cell elongation. Parallel actin bundles (PABs) are usually cross-linked by more than one actin-bundling protein. Accordingly, we have also shown that sn synergistically interacts with forked (f), another actin crosslinker. In this Extra View we extend f analysis and hypothesize how both actin-bundling proteins may act together to regulate the PABs during tracheal embryonic development. Although both proteins are required for similar tracheal events, we suggest that Sn is essential for actin bundle initiation and stiffening, while F is required for the lengthening and further stabilization of the PABs.     

Actomyosin-dependent cortical dynamics contributes to the prophase force-balance in the early Drosophila embryo

Background

The assembly of the Drosophila embryo mitotic spindle during prophase depends upon a balance of outward forces generated by cortical dynein and inward forces generated by kinesin-14 and nuclear elasticity. Myosin II is known to contribute to the dynamics of the cell cortex but how this influences the prophase force-balance is unclear.

Principal Findings

Here we investigated this question by injecting the myosin II inhibitor, Y27632, into early Drosophila embryos. We observed a significant increase in both the area of the dense cortical actin caps and in the spacing of the spindle poles. Tracking of microtubule plus ends marked by EB1-GFP and of actin at the cortex revealed that astral microtubules can interact with all regions of these expanded caps, presumably via their interaction with cortical dynein. In Scrambled mutants displaying abnormally small actin caps but normal prophase spindle length in late prophase, myosin II inhibition produced very short spindles.

Conclusions

These results suggest that two complementary outward forces are exerted on the prophase spindle by the overlying cortex. Specifically, dynein localized on the mechanically firm actin caps and the actomyosin-driven contraction of the deformable soft patches of the actin cortex, cooperate to pull astral microtubules outward. Thus, myosin II controls the size and dynamic properties of the actin-based cortex to influence the spacing of the poles of the underlying spindle during prophase.     

Diaphanous regulates SCAR complex localization during Drosophila myoblast fusion

From Drosophila to man, multinucleated muscle cells form through cell-cell fusion. Using Drosophila as a model system, researchers first identified, and then demonstrated, the importance of actin cytoskeletal rearrangements at the site of fusion. These actin rearrangements at the fusion site are regulated by SCAR and WASp mediated Arp2/3 activation, which nucleates branched actin networks. Loss of SCAR, WASp or both leads to defects in myoblast fusion. Recently, we have found that the actin regulator Diaphanous (Dia) also plays a role both in organizing actin and in regulating Arp2/3 activity at the fusion site. In this Extra View article, we provide additional data showing that the Abi-SCAR complex accumulates at the fusion site and that excessive SCAR activity impairs myoblast fusion. Using constitutively active Dia constructs, we provide additional evidence that Dia functions upstream of SCAR activity to regulate actin dynamics at the fusion site and to localize the Abi-SCAR complex.     

Fluctuation Analysis of Centrosomes Reveals a Cortical Function of Kinesin-1

The actin and microtubule networks form the dynamic cytoskeleton. Network dynamics is driven by molecular motors applying force onto the networks and the interactions between the networks. Here we assay the dynamics of centrosomes in the scale of seconds as a proxy for the movement of microtubule asters. With this assay we want to detect the role of specific motors and of network interaction. During interphase of syncytial embryos of Drosophila, cortical actin and the microtubule network depend on each other. Centrosomes induce cortical actin to form caps, whereas F-actin anchors microtubules to the cortex. In addition, lateral interactions between microtubule asters are assumed to be important for regular spatial organization of the syncytial embryo. The functional interaction between the microtubule asters and cortical actin has been largely analyzed in a static manner, so far. We recorded the movement of centrosomes at 1 Hz and analyzed their fluctuations for two processes—pair separation and individual movement. We found that F-actin is required for directional movements during initial centrosome pair separation, because separation proceeds in a diffusive manner in latrunculin-injected embryos. For assaying individual movement, we established a fluctuation parameter as the deviation from temporally and spatially slowly varying drift movements. By analysis of mutant and drug-injected embryos, we found that the fluctuations were suppressed by both cortical actin and microtubules. Surprisingly, the microtubule motor Kinesin-1 also suppressed fluctuations to a similar degree as F-actin. Kinesin-1 may mediate linkage of the microtubule (+)-ends to the actin cortex. Consistent with this model is our finding that Kinesin-1-GFP accumulates at the cortical actin caps.     

Dynamin-2 Regulates Fusion Pore Expansion and Quantal Release through a Mechanism that Involves Actin Dynamics in Neuroendocrine Chromaffin Cells

Over the past years, dynamin has been implicated in tuning the amount and nature of transmitter released during exocytosis. However, the mechanism involved remains poorly understood. Here, using bovine adrenal chromaffin cells, we investigated whether this mechanism rely on dynamin’s ability to remodel actin cytoskeleton. According to this idea, inhibition of dynamin GTPase activity suppressed the calcium-dependent de novo cortical actin and altered the cortical actin network. Similarly, expression of a small interfering RNA directed against dynamin-2, an isoform highly expressed in chromaffin cells, changed the cortical actin network pattern. Disruption of dynamin-2 function, as well as the pharmacological inhibition of actin polymerization with cytochalasine-D, slowed down fusion pore expansion and increased the quantal size of individual exocytotic events. The effects of cytochalasine-D and dynamin-2 disruption were not additive indicating that dynamin-2 and F-actin regulate the late steps of exocytosis by a common mechanism. Together our data support a model in which dynamin-2 directs actin polymerization at the exocytosis site where both, in concert, adjust the hormone quantal release to efficiently respond to physiological demands.     

A novel Drosophila Girdin-like protein is involved in Akt pathway control of cell size

The Akt signaling pathway is well known to regulate cell proliferation and growth. Girdin, a novel substrate of Akt, plays a crucial role in organization of the actin cytoskeleton and cell motility under the control of Akt. We here identified a novel Girdin-like protein in Drosophila (dGirdin), which has two isoforms, dGirdin PA and dGirdin PB. dGirdin shows high homology with human Girdin in the N-terminal and coiled-coil domains, while diverging at the C-terminal domain. On establishment of transgenic fly lines, featuring knockdown or overexpression of dGirdin in vivo, overexpression in the wing disc cells induced ectopic apoptosis, implying a role in directing apoptosis. Knockdown of dGirdin in the Drosophila wing imaginal disc cells resulted in reduction of cell size. Furthermore, this was enhanced by half reduction of the Akt gene dose, suggesting that Akt positively regulates dGirdin. In the wing disc, cells in which dGirdin was knocked down exhibited disruption of actin filaments. From these in vivo analyses, we conclude that dGirdin is required for actin organization and regulation of appropriate cell size under control of the Akt signaling pathway.     

Kank Is an EB1 Interacting Protein that Localises to Muscle-Tendon Attachment Sites in Drosophila

Little is known about how microtubules are regulated in different cell types during development. EB1 plays a central role in the regulation of microtubule plus ends. It directly binds to microtubule plus ends and recruits proteins which regulate microtubule dynamics and behaviour. We report the identification of Kank, the sole Drosophila orthologue of human Kank proteins, as an EB1 interactor that predominantly localises to embryonic attachment sites between muscle and tendon cells. Human Kank1 was identified as a tumour suppressor and has documented roles in actin regulation and cell polarity in cultured mammalian cells. We found that Drosophila Kank binds EB1 directly and this interaction is essential for Kank localisation to microtubule plus ends in cultured cells. Kank protein is expressed throughout fly development and increases during embryogenesis. In late embryos, it accumulates to sites of attachment between muscle and epidermal cells. A kank deletion mutant was generated. We found that the mutant is viable and fertile without noticeable defects. Further analysis showed that Kank is dispensable for muscle function in larvae. This is in sharp contrast to C. elegans in which the Kank orthologue VAB-19 is required for development by stabilising attachment structures between muscle and epidermal cells.     

TAGLN2 regulates T cell activation by stabilizing the actin cytoskeleton at the immunological synapse

The formation of an immunological synapse (IS) requires tight regulation of actin dynamics by many actin polymerizing/depolymerizing proteins. However, the significance of actin stabilization at the IS remains largely unknown. In this paper, we identify a novel function of TAGLN2—an actin-binding protein predominantly expressed in T cells—in stabilizing cortical F-actin, thereby maintaining F-actin contents at the IS and acquiring LFA-1 (leukocyte function-associated antigen-1) activation after T cell receptor stimulation. TAGLN2 blocks actin depolymerization and competes with cofilin both in vitro and in vivo. Knockout of TAGLN2 (TAGLN2^−/−) reduced F-actin content and destabilized F-actin ring formation, resulting in decreased cell adhesion and spreading. TAGLN2^−/− T cells displayed weakened cytokine production and cytotoxic effector function. These findings reveal a novel function of TAGLN2 in enhancing T cell responses by controlling actin stability at the IS.