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.     

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.     

Live Imaging Provides New Insights on Dynamic F-Actin Filopodia and Differential Endocytosis during Myoblast Fusion in Drosophila

The process of myogenesis includes the recognition, adhesion, and fusion of committed myoblasts into multinucleate syncytia. In the larval body wall muscles of Drosophila, this elaborate process is initiated by Founder Cells and Fusion-Competent Myoblasts (FCMs), and cell adhesion molecules Kin-of-IrreC (Kirre) and Sticks-and-stones (Sns) on their respective surfaces. The FCMs appear to provide the driving force for fusion, via the assembly of protrusions associated with branched F-actin and the WASp, SCAR and Arp2/3 pathways. In the present study, we utilize the dorsal pharyngeal musculature that forms in the Drosophila embryo as a model to explore myoblast fusion and visualize the fusion process in live embryos. These muscles rely on the same cell types and genes as the body wall muscles, but are amenable to live imaging since they do not undergo extensive morphogenetic movement during formation. Time-lapse imaging with F-actin and membrane markers revealed dynamic FCM-associated actin-enriched protrusions that rapidly extend and retract into the myotube from different sites within the actin focus. Ultrastructural analysis of this actin-enriched area showed that they have two morphologically distinct structures: wider invasions and/or narrow filopodia that contain long linear filaments. Consistent with this, formin Diaphanous (Dia) and branched actin nucleator, Arp3, are found decorating the filopodia or enriched at the actin focus, respectively, indicating that linear actin is present along with branched actin at sites of fusion in the FCM. Gain-of-function Dia and loss-of-function Arp3 both lead to fusion defects, a decrease of F-actin foci and prominent filopodia from the FCMs. We also observed differential endocytosis of cell surface components at sites of fusion, with actin reorganizing factors, WASp and SCAR, and Kirre remaining on the myotube surface and Sns preferentially taken up with other membrane proteins into early endosomes and lysosomes in the myotube.     

The Formin Diaphanous Regulates Myoblast Fusion through Actin Polymerization and Arp2/3 Regulation

The formation of multinucleated muscle cells through cell-cell fusion is a conserved process from fruit flies to humans. Numerous studies have shown the importance of Arp2/3, its regulators, and branched actin for the formation of an actin structure, the F-actin focus, at the fusion site. This F-actin focus forms the core of an invasive podosome-like structure that is required for myoblast fusion. In this study, we find that the formin Diaphanous (Dia), which nucleates and facilitates the elongation of actin filaments, is essential for Drosophila myoblast fusion. Following cell recognition and adhesion, Dia is enriched at the myoblast fusion site, concomitant with, and having the same dynamics as, the F-actin focus. Through analysis of Dia loss-of-function conditions using mutant alleles but particularly a dominant negative Dia transgene, we demonstrate that reduction in Dia activity in myoblasts leads to a fusion block. Significantly, no actin focus is detected, and neither branched actin regulators, SCAR or WASp, accumulate at the fusion site when Dia levels are reduced. Expression of constitutively active Dia also causes a fusion block that is associated with an increase in highly dynamic filopodia, altered actin turnover rates and F-actin distribution, and mislocalization of SCAR and WASp at the fusion site. Together our data indicate that Dia plays two roles during invasive podosome formation at the fusion site: it dictates the level of linear F-actin polymerization, and it is required for appropriate branched actin polymerization via localization of SCAR and WASp. These studies provide new insight to the mechanisms of cell-cell fusion, the relationship between different regulators of actin polymerization, and invasive podosome formation that occurs in normal development and in disease.     

Drosophila Fascin is a novel downstream target of prostaglandin signaling during actin remodeling

Although prostaglandins (PGs)—lipid signals produced downstream of cyclooxygenase (COX) enzymes—regulate actin cytoskeletal dynamics, their mechanisms of action are unknown. We previously established Drosophila oogenesis, in particular nurse cell dumping, as a new model to determine how PGs regulate actin remodeling. PGs, and thus the Drosophila COX-like enzyme Pxt, are required for both the parallel actin filament bundle formation and the cortical actin strengthening required for dumping. Here we provide the first link between Fascin (Drosophila Singed, Sn), an actin-bundling protein, and PGs. Loss of either pxt or fascin results in similar actin defects. Fascin interacts, both pharmacologically and genetically, with PGs, as reduced Fascin levels enhance the effects of COX inhibition and synergize with reduced Pxt levels to cause both parallel bundle and cortical actin defects. Conversely, overexpression of Fascin in the germline suppresses the effects of COX inhibition and genetic loss of Pxt. These data lead to the conclusion that PGs regulate Fascin to control actin remodeling. This novel interaction has implications beyond Drosophila, as both PGs and Fascin-1, in mammalian systems, contribute to cancer cell migration and invasion.     

The role of tropomyosin in the interactions of F-actin with caldesmon and actin-binding protein (or filamin)

The interactions of actin filaments with actin-binding protein (filamin) and caldesmon under the influence of tropomyosin were studied in detail using falling-ball viscometry, binding assay and electron microscopy. Caldesmon decreased the binding constant of filamin with F-actin. In contrast, the maximum binding ability of filamin to F-actin was decreased by tropomyosin. The filamin-induced gelation of actin filaments was inhibited by caldesmon. Tropomyosin also inhibited this gelation. The effect of caldesmon became stronger under the influence of tropomyosin. Furthermore, both caldesmon and tropomyosin additionally decreased the filamin binding to F-actin. From these results, caldesmon and tropomyosin appeared to influence filamin binding to F-actin with different modes of actin. In addition, there was no sign of direct interactions between filamin, caldesmon and tropomyosin as judged from gel filtration. Under the influence of caldesmon and tropomyosin, calmodulin conferred Ca2+ sensitivity on the filamin-induced gelation of actin filaments.     

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.     

Impaired macrophage autophagy increases the immune response in obese mice by promoting proinflammatory macrophage polarization

Recent evidence that excessive lipid accumulation can decrease cellular levels of autophagy and that autophagy regulates immune responsiveness suggested that impaired macrophage autophagy may promote the increased innate immune activation that underlies obesity. Primary bone marrow-derived macrophages (BMDM) and peritoneal macrophages from high-fat diet (HFD)-fed mice had decreased levels of autophagic flux indicating a generalized impairment of macrophage autophagy in obese mice. To assess the effects of decreased macrophage autophagy on inflammation, mice with a Lyz2-Cre-mediated knockout of Atg5 in macrophages were fed a HFD and treated with low-dose lipopolysaccharide (LPS). Knockout mice developed systemic and hepatic inflammation with HFD feeding and LPS. This effect was liver specific as knockout mice did not have increased adipose tissue inflammation. The mechanism by which the loss of autophagy promoted inflammation was through the regulation of macrophage polarization. BMDM and Kupffer cells from knockout mice exhibited abnormalities in polarization with both increased proinflammatory M1 and decreased anti-inflammatory M2 polarization as determined by measures of genes and proteins. The heightened hepatic inflammatory response in HFD-fed, LPS-treated knockout mice led to liver injury without affecting steatosis. These findings demonstrate that autophagy has a critical regulatory function in macrophage polarization that downregulates inflammation. Defects in macrophage autophagy may underlie inflammatory disease states such as the decrease in macrophage autophagy with obesity that leads to hepatic inflammation and the progression to liver injury.     

Microbial sphingomyelinase induces RhoA-mediated reorganization of the apical brush border membrane and is protective against invasion

The apical brush border membrane (BBM) of intestinal epithelial cells forms a highly structured and dynamic environmental interface that serves to regulate cellular physiology and block invasion by intestinal microbes and their products. How the BBM dynamically responds to pathogenic and commensal bacterial signals can define intestinal homeostasis and immune function. We previously found that in model intestinal epithelium, the conversion of apical membrane sphingomyelin to ceramide by exogenous bacterial sphingomyelinase (SMase) protected against the endocytosis and toxicity of cholera toxin. Here we elucidate a mechanism of action by showing that SMase induces a dramatic, reversible, RhoA-dependent alteration of the apical cortical F-actin network. Accumulation of apical membrane ceramide is necessary and sufficient to induce the actin phenotype, and this coincides with altered membrane structure and augmented innate immune function as evidenced by resistance to invasion by Salmonella.     

The bimodal role of filamin in controlling the architecture and mechanics of F-actin networks

Reconstituted actin filament networks have been used extensively to understand the mechanics of the actin cortex and decipher the role of actin cross-linking proteins in the maintenance and deformation of cell shape. However, studies of the mechanical role of the F-actin cross-linking protein filamin have led to seemingly contradictory conclusions, in part due to the use of ill-defined mechanical assays. Using quantitative rheological methods that avoid the pitfalls of previous studies, we systematically tested the complex mechanical response of reconstituted actin filament networks containing a wide range of filamin concentrations and compared the mechanical function of filamin with that of the cross-linking/bundling proteins alpha-actinin and fascin. At steady state and within a well defined linear regime of small non-destructive deformations, F-actin solutions behave as highly dynamic networks (actin polymers are still sufficiently mobile to relax the stress) below the cross-linking-to-bundling threshold filamin concentration, and they behave as covalently cross-linked gels above that threshold. Under large deformations, F-actin networks soften at low filamin concentrations and strain-harden at high filamin concentrations. Filamin cross-links F-actin into networks that are more resilient, stiffer, more solid-like, and less dynamic than alpha-actinin and fascin. These results resolve the controversy by showing that F-actin/filamin networks can adopt diametrically opposed rheological behaviors depending on the concentration in cross-linking proteins.     

Src controls tumorigenesis via JNK‐dependent regulation of the Hippo pathway in Drosophila

Cell–cell interactions within the tumour microenvironment have crucial roles in epithelial tumorigenesis. Using Drosophila genetics, we show that the oncoprotein Src controls tumour microenvironment by Jun N‐terminal kinase (JNK)‐dependent regulation of the Hippo pathway. Clones of cells with elevated Src expression activate the Rac‐Diaphanous and Ras‐mitogen‐activated protein kinase (MAPK) pathways, which cooperatively induce F‐actin accumulation, thereby leading to activation of the Hippo pathway effector Yorkie (Yki). Simultaneously, Src activates the JNK pathway, which antagonizes the autonomous Yki activity and causes propagation of Yki activity to neighbouring cells, resulting in the overgrowth of surrounding tissue. Our data provide a mechanism to explain how oncogenic mutations regulate tumour microenvironment through cell–cell communication.     

Small-molecule agonists of mammalian Diaphanous–related (mDia) formins reveal an effective glioblastoma anti-invasion strategy

The extensive invasive capacity of glioblastoma (GBM) makes it resistant to surgery, radiotherapy, and chemotherapy and thus makes it lethal. In vivo, GBM invasion is mediated by Rho GTPases through unidentified downstream effectors. Mammalian Diaphanous (mDia) family formins are Rho-directed effectors that regulate the F-actin cytoskeleton to support tumor cell motility. Historically, anti-invasion strategies focused upon mDia inhibition, whereas activation remained unexplored. The recent development of small molecules directly inhibiting or activating mDia-driven F-actin assembly that supports motility allows for exploration of their role in GBM. We used the formin inhibitor SMIFH2 and mDia agonists IMM-01/-02 and mDia2-DAD peptides, which disrupt autoinhibition, to examine the roles of mDia inactivation versus activation in GBM cell migration and invasion in vitro and in an ex vivo brain slice invasion model. Inhibiting mDia suppressed directional migration and spheroid invasion while preserving intrinsic random migration. mDia agonism abrogated both random intrinsic and directional migration and halted U87 spheroid invasion in ex vivo brain slices. Thus mDia agonism is a superior GBM anti-invasion strategy. We conclude that formin agonism impedes the most dangerous GBM component—tumor spread into surrounding healthy tissue. Formin activation impairs novel aspects of transformed cells and informs the development of anti-GBM invasion strategies.     

α-Actinin and Filamin Cooperatively Enhance the Stiffness of Actin Filament Networks

Background

The close subcellular proximity of different actin filament crosslinking proteins suggests that these proteins may cooperate to organize F-actin structures to drive complex cellular functions during cell adhesion, motility and division. Here we hypothesize that α-actinin and filamin, two major F-actin crosslinking proteins that are both present in the lamella of adherent cells, display synergistic mechanical functions.

Methodology/Principal Findings

Using quantitative rheology, we find that combining α-actinin and filamin is much more effective at producing elastic, solid-like actin filament networks than α-actinin and filamin separately. Moreover, F-actin networks assembled in the presence of α-actinin and filamin strain-harden more readily than networks in the presence of either α-actinin or filamin.

Significance

These results suggest that cells combine auxiliary proteins with similar ability to crosslink filaments to generate stiff cytoskeletal structures, which are required for the production of internal propulsive forces for cell migration, and that these proteins do not have redundant mechanical functions.     

Drosophila Kelch functions with Cullin-3 to organize the ring canal actin cytoskeleton

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.     

Interaction of alpha-actinin, filamin and tropomyosin with F-actin

The abilities of alpha-actinin, filamin and tropomyosin to bind F-actin were examined by cosedimentation experiments. Results indicated that smooth muscle alpha-actinin and filamin can bind to actin filaments simultaneously with little evidence of competition. In contrast, tropomyosin exhibits marked competition with either filamin or alpha-actinin for sites on actin filaments.     

Inositol phospholipid-induced suppression of F-actin-gelating activity of smooth muscle filamin.

Filamin, a high molecular weight actin-binding protein, cross-links actin filaments and produces a gel composed of F-actin. The effects of polyphosphoinositides on the gelating activity of smooth muscle filamin were examined by measuring the low shear viscosity of the F-actin solutions containing filamin incubated with phosphatidylinositol (PI), phosphatidylinositol 4-monophosphate (PIP), or phosphatidylinositol 4,5-bisphosphate (PIP2). Micelles of these inositol phospholipids bound to filamin inhibited the ability to form a gel of F-actin. The inhibiting activity of each phospholipid was in the following order, PIP2 greater than PIP greater than PI. The F-actin binding assay of filamin revealed that the inhibition of F-actin-gelation resulted in the loss of the F-actin-binding activity of filamin. Thus, polyphosphoinositides may play important roles in regulating the gelating activity of filamin.     

The PCH family member MAYP/PSTPIP2 directly regulates F-actin bundling and enhances filopodia formation and motility in macrophages

Macrophage actin-associated tyrosine phosphorylated protein (MAYP) belongs to the Pombe Cdc15 homology (PCH) family of proteins involved in the regulation of actin-based functions including cell adhesion and motility. In mouse macrophages, MAYP is tyrosine phosphorylated after activation of the colony-stimulating factor-1 receptor (CSF-1R), which also induces actin reorganization, membrane ruffling, cell spreading, polarization, and migration. Because MAYP associates with F-actin, we investigated the function of MAYP in regulating actin organization in macrophages. Overexpression of MAYP decreased CSF-1-induced membrane ruffling and increased filopodia formation, motility and CSF-1-mediated chemotaxis. The opposite phenotype was observed with reduced expression of MAYP, indicating that MAYP is a negative regulator of CSF-1-induced membrane ruffling and positively regulates formation of filopodia and directional migration. Overexpression of MAYP led to a reduction in total macrophage F-actin content but was associated with increased actin bundling. Consistent with this, purified MAYP bundled F-actin and regulated its turnover in vitro. In addition, MAYP colocalized with cortical and filopodial F-actin in vivo. Because filopodia are postulated to increase directional motility by acting as environmental sensors, the MAYP-stimulated increase in directional movement may be at least partly explained by enhancement of filopodia formation.     

Drosophila cyfip Regulates Synaptic Development and Endocytosis by Suppressing Filamentous Actin Assembly

The formation of synapses and the proper construction of neural circuits depend on signaling pathways that regulate cytoskeletal structure and dynamics. After the mutual recognition of a growing axon and its target, multiple signaling pathways are activated that regulate cytoskeletal dynamics to determine the morphology and strength of the connection. By analyzing Drosophila mutations in the cytoplasmic FMRP interacting protein Cyfip, we demonstrate that this component of the WAVE complex inhibits the assembly of filamentous actin (F-actin) and thereby regulates key aspects of synaptogenesis. Cyfip regulates the distribution of F-actin filaments in presynaptic neuromuscular junction (NMJ) terminals. At cyfip mutant NMJs, F-actin assembly was accelerated, resulting in shorter NMJs, more numerous satellite boutons, and reduced quantal content. Increased synaptic vesicle size and failure to maintain excitatory junctional potential amplitudes under high-frequency stimulation in cyfip mutants indicated an endocytic defect. cyfip mutants exhibited upregulated bone morphogenetic protein (BMP) signaling, a major growth-promoting pathway known to be attenuated by endocytosis at the Drosophila NMJ. We propose that Cyfip regulates synapse development and endocytosis by inhibiting actin assembly.     

Group I PAKs function downstream of Rac to promote podosome invasion during myoblast fusion in vivo

The p21-activated kinases (PAKs) play essential roles in diverse cellular processes and are required for cell proliferation, apoptosis, polarity establishment, migration, and cell shape changes. Here, we have identified a novel function for the group I PAKs in cell–cell fusion. We show that the two Drosophila group I PAKs, DPak3 and DPak1, have partially redundant functions in myoblast fusion in vivo, with DPak3 playing a major role. DPak3 is enriched at the site of fusion colocalizing with the F-actin focus within a podosome-like structure (PLS), and promotes actin filament assembly during PLS invasion. Although the small GTPase Rac is involved in DPak3 activation and recruitment to the PLS, the kinase activity of DPak3 is required for effective PLS invasion. We propose a model whereby group I PAKs act downstream of Rac to organize the actin filaments within the PLS into a dense focus, which in turn promotes PLS invasion and fusion pore initiation during myoblast fusion.     

The WAVE/SCAR complex promotes polarized cell movements and actin enrichment in epithelia during C. elegans embryogenesis

The WAVE/SCAR complex promotes actin nucleation through the Arp2/3 complex, in response to Rac signaling. We show that loss of WVE-1/GEX-1, the only C. elegans WAVE/SCAR homolog, by genetic mutation or by RNAi, has the same phenotype as loss of GEX-2/Sra1/p140/PIR121, GEX-3/NAP1/HEM2/KETTE, or ABI-1/ABI, the three other components of the C. elegans WAVE/SCAR complex. We find that the entire WAVE/SCAR complex promotes actin-dependent events at different times and in different tissues during development. During C. elegans embryogenesis loss of CED-10/Rac1, WAVE/SCAR complex components, or Arp2/3 blocks epidermal cell migrations despite correct epidermal cell differentiation. 4D movies show that this failure occurs due to decreased membrane dynamics in specific epidermal cells. Unlike myoblasts in Drosophila, epidermal cell fusions in C. elegans can occur in the absence of WAVE/SCAR or Arp2/3. Instead we find that subcellular enrichment of F-actin in epithelial tissues requires the Rac-WAVE/SCAR-Arp2/3 pathway. Intriguingly, we find that at the same stage of development both F-actin and WAVE/SCAR proteins are enriched apically in one epithelial tissue and basolaterally in another. We propose that temporally and spatially regulated actin nucleation by the Rac-WAVE/SCAR-Arp2/3 pathway is required for epithelial cell organization and movements during morphogenesis.