CryoET shows cofilactin filaments inside the microtubule lumen.

Cytoplasmic microtubules are tubular polymers that can harbor small proteins or filaments inside their lumen. The identities of these objects and mechanisms for their accumulation have not been conclusively established. Here, we used cryogenic electron tomography of Drosophila S2 cell protrusions and found filaments inside the microtubule lumen, which resemble those reported recently in human HAP1 cells. The frequency of these filaments increased upon inhibition of the sarco/endoplasmic reticulum Ca2+ ATPase with the small molecule drug thapsigargin. Subtomogram averaging showed that the luminal filaments adopt a helical structure reminiscent of cofilin-bound actin (cofilactin). Consistent with this, we observed cofilin dephosphorylation, an activating modification, in cells under the same conditions that increased luminal filament occurrence. Furthermore, RNA interference knock-down of cofilin reduced the frequency of luminal filaments with cofilactin morphology. These results suggest that cofilin activation stimulates its accumulation on actin filaments inside the microtubule lumen.

CryoET shows cofilactin filaments inside the microtubule lumen.

Cytoplasmic microtubules are tubular polymers that can harbor small proteins or filaments inside their lumen. The identity of these objects and what causes their accumulation has not been conclusively established. Here, we used cryogenic electron tomography (cryoET) of Drosophila S2 cell protrusions and found filaments inside the microtubule lumen, which resemble those reported recently in human HAP1 cells. The frequency of these filaments increased upon inhibition of the sarco/endoplasmic reticulum Ca 2+ ATPase (SERCA) with the small-molecule drug thapsigargin. Subtomogram averaging showed that the luminal filaments adopt a helical structure reminiscent of cofilin-bound actin (cofilactin). Consistent with this, cofilin was activated in cells under the same conditions that increased luminal filament occurrence. Furthermore, RNAi knock-down of cofilin reduced the frequency of luminal filaments with cofilactin morphology. These results suggest that cofilin activation stimulates its accumulation on actin filaments inside the microtubule lumen.

The Drosophila spectraplakin Short stop regulates focal adhesion dynamics by cross-linking microtubules and actin.

The spectraplakin family of proteins includes ACF7/MACF1 and BPAG1/dystonin in mammals, VAB-10 in Caenorhabditis elegans, Magellan in zebrafish, and Short stop (Shot), the sole Drosophila member. Spectraplakins are giant cytoskeletal proteins that cross-link actin, microtubules, and intermediate filaments, coordinating the activity of the entire cytoskeleton. We examined the role of Shot during cell migration using two systems: the in vitro migration of Drosophila tissue culture cells and in vivo through border cell migration. RNA interference (RNAi) depletion of Shot increases the rate of random cell migration in Drosophila tissue culture cells as well as the rate of wound closure during scratch-wound assays. This increase in cell migration prompted us to analyze focal adhesion dynamics. We found that the rates of focal adhesion assembly and disassembly were faster in Shot-depleted cells, leading to faster adhesion turnover that could underlie the increased migration speeds. This regulation of focal adhesion dynamics may be dependent on Shot being in an open confirmation. Using Drosophila border cells as an in vivo model for cell migration, we found that RNAi depletion led to precocious border cell migration. Collectively, these results suggest that spectraplakins not only function to cross-link the cytoskeleton but may regulate cell-matrix adhesion.

Serum-free adapted Drosophila S2R+ line is amenable to RNA interference.

We have previously adapted a select number of Drosophila cell lines to grow in serum-free media supplemented with fly extract. This condition is arguably more representative of a native growth environment. Here, we validated that the fly extract adapted line, S2R+ (FEx 2.5%) is amenable to RNAi. RNAi against Rho1 in both S2R+ and S2R+ (FEx 2.5%) produced phenotypes similar to ones previously described in Drosophila S2 cells.

The Drosophila melanogaster Rab GAP RN-tre cross-talks with the Rho1 signaling pathway to regulate nonmuscle myosin II localization and function.

To identify novel regulators of nonmuscle myosin II (NMII) we performed an image-based RNA interference screen using stable Drosophila melanogaster S2 cells expressing the enhanced green fluorescent protein (EGFP)-tagged regulatory light chain (RLC) of NMII and mCherry-Actin. We identified the Rab-specific GTPase-activating protein (GAP) RN-tre as necessary for the assembly of NMII RLC into contractile actin networks. Depletion of RN-tre led to a punctate NMII phenotype, similar to what is observed following depletion of proteins in the Rho1 pathway. Depletion of RN-tre also led to a decrease in active Rho1 and a decrease in phosphomyosin-positive cells by immunostaining, while expression of constitutively active Rho or Rho-kinase (Rok) rescues the punctate phenotype. Functionally, RN-tre depletion led to an increase in actin retrograde flow rate and cellular contractility in S2 and S2R+ cells, respectively. Regulation of NMII by RN-tre is only partially dependent on its GAP activity as overexpression of constitutively active Rabs inactivated by RN-tre failed to alter NMII RLC localization, while a GAP-dead version of RN-tre partially restored phosphomyosin staining. Collectively, our results suggest that RN-tre plays an important regulatory role in NMII RLC distribution, phosphorylation, and function, likely through Rho1 signaling and putatively serving as a link between the secretion machinery and actomyosin contractility.

Non-enzymatic Activity of the α-Tubulin Acetyltransferase αTAT Limits Synaptic Bouton Growth in Neurons.

Neuronal axons terminate as synaptic boutons that form stable yet plastic connections with their targets. Synaptic bouton development relies on an underlying network of both long-lived and dynamic microtubules that provide structural stability for the boutons while also allowing for their growth and remodeling. However, a molecular-scale mechanism that explains how neurons appropriately balance these two microtubule populations remains a mystery. We hypothesized that α-tubulin acetyltransferase (αTAT), which both stabilizes long-lived microtubules against mechanical stress via acetylation and has been implicated in promoting microtubule dynamics, could play a role in this process. Using the Drosophila neuromuscular junction as a model, we found that non-enzymatic dαTAT activity limits the growth of synaptic boutons by affecting dynamic, but not stable, microtubules. Loss of dαTAT results in the formation of ectopic boutons. These ectopic boutons can be similarly suppressed by resupplying enzyme-inactive dαTAT or by treatment with a low concentration of the microtubule-targeting agent vinblastine, which acts to suppress microtubule dynamics. Biophysical reconstitution experiments revealed that non-enzymatic αTAT1 activity destabilizes dynamic microtubules but does not substantially impact the stability of long-lived microtubules. Further, during microtubule growth, non-enzymatic αTAT1 activity results in increasingly extended tip structures, consistent with an increased rate of acceleration of catastrophe frequency with microtubule age, perhaps via tip structure remodeling. Through these mechanisms, αTAT enriches for stable microtubules at the expense of dynamic ones. We propose that the specific suppression of dynamic microtubules by non-enzymatic αTAT activity regulates the remodeling of microtubule networks during synaptic bouton development.

Microtubule Acetylation Is Required for Mechanosensation in Drosophila.

At the cellular level, α-tubulin acetylation alters the structure of microtubules to render them mechanically resistant to compressive forces. How this biochemical property of microtubule acetylation relates to mechanosensation remains unknown, although prior studies have shown that microtubule acetylation influences touch perception. Here, we identify the major Drosophila α-tubulin acetylase (dTAT) and show that it plays key roles in several forms of mechanosensation. dTAT is highly expressed in the larval peripheral nervous system (PNS), but it is largely dispensable for neuronal morphogenesis. Mutation of the acetylase gene or the K40 acetylation site in α-tubulin impairs mechanical sensitivity in sensory neurons and behavioral responses to gentle touch, harsh touch, gravity, and vibration stimuli, but not noxious thermal stimulus. Finally, we show that dTAT is required for mechanically induced activation of NOMPC, a microtubule-associated transient receptor potential channel, and functions to maintain integrity of the microtubule cytoskeleton in response to mechanical stimulation.

A Cell-based Assay to Investigate Non-muscle Myosin II Contractility via the Folded-gastrulation Signaling Pathway in Drosophila S2R+ Cells.

We have developed a cell-based assay using Drosophila cells that recapitulates apical constriction initiated by folded gastrulation (Fog), a secreted epithelial morphogen. In this assay, Fog is used as an agonist to activate Rho through a signaling cascade that includes a G-protein-coupled receptor (Mist), a Gα12/13 protein (Concertina/Cta), and a PDZ-domain-containing guanine nucleotide exchange factor (RhoGEF2). Fog signaling results in the rapid and dramatic reorganization of the actin cytoskeleton to form a contractile purse string. Soluble Fog is collected from a stable cell line and applied ectopically to S2R+ cells, leading to morphological changes like apical constriction, a process observed during developmental processes such as gastrulation. This assay is amenable to high-throughput screening and, using RNAi, can facilitate the identification of additional genes involved in this pathway.

Drosophila Ringmaker regulates microtubule stabilization and axonal extension during embryonic development.

Axonal growth and targeting are fundamental to the organization of the nervous system, and require active engagement of the cytoskeleton. Polymerization and stabilization of axonal microtubules is central to axonal growth and maturation of neuronal connectivity. Studies have suggested that members of the tubulin polymerization promoting protein (TPPP, also known as P25α) family are involved in cellular process extension. However, no in vivo knockout data exists regarding its role in axonal growth during development. Here, we report the characterization of Ringmaker (Ringer; CG45057), the only Drosophila homolog of long p25α proteins. Immunohistochemical analyses indicate that Ringer expression is dynamically regulated in the embryonic central nervous system (CNS). ringer-null mutants show cell misplacement, and errors in axonal extension and targeting. Ultrastructural examination of ringer mutants revealed defective microtubule morphology and organization. Primary neuronal cultures of ringer mutants exhibit defective axonal extension, and Ringer expression in cells induced microtubule stabilization and bundling into rings. In vitro assays showed that Ringer directly affects tubulin, and promotes microtubule bundling and polymerization. Together, our studies uncover an essential function of Ringer in axonal extension and targeting through proper microtubule organization.

The Gas2 family protein Pigs is a microtubule +TIP that affects cytoskeleton organisation.

Coordination between different cytoskeletal systems is crucial for many cell biological functions, including cell migration and mitosis, and also plays an important role during tissue morphogenesis. Proteins of the class of cytoskeletal crosslinkers, or cytolinkers, have the ability to interact with more than one cytoskeletal system at a time and are prime candidates to mediate any coordination. One such class comprises the Gas2-like proteins, combining a conserved calponin-homology-type actin-binding domain and a Gas2 domain predicted to bind microtubules (MTs). This domain combination is also found in spectraplakins, huge cytolinkers that play important roles in many tissues in both invertebrates and vertebrates. Here, we dissect the ability of the single Drosophila Gas2-like protein Pigs to interact with both actin and MT cytoskeletons, both in vitro and in vivo, and illustrate complex regulatory interactions that determine the localisation of Pigs to and its effects on the cytoskeleton.

TOG Proteins Are Spatially Regulated by Rac-GSK3ß to Control Interphase Microtubule Dynamics.

Microtubules are regulated by a diverse set of proteins that localize to microtubule plus ends (+TIPs) where they regulate dynamic instability and mediate interactions with the cell cortex, actin filaments, and organelles. Although individual +TIPs have been studied in depth and we understand their basic contributions to microtubule dynamics, there is a growing body of evidence that these proteins exhibit cross-talk and likely function to collectively integrate microtubule behavior and upstream signaling pathways. In this study, we have identified a novel protein-protein interaction between the XMAP215 homologue in Drosophila, Mini spindles (Msps), and the CLASP homologue, Orbit. These proteins have been shown to promote and suppress microtubule dynamics, respectively. We show that microtubule dynamics are regionally controlled in cells by Rac acting to suppress GSK3ß in the peripheral lamellae/lamellipodium. Phosphorylation of Orbit by GSK3ß triggers a relocalization of Msps from the microtubule plus end to the lattice. Mutation of the Msps-Orbit binding site revealed that this interaction is required for regulating microtubule dynamic instability in the cell periphery. Based on our findings, we propose that Msps is a novel Rac effector that acts, in partnership with Orbit, to regionally regulate microtubule dynamics.

The non-catalytic domains of Drosophila katanin regulate its abundance and microtubule-disassembly activity.

Microtubule severing is a biochemical reaction that generates an internal break in a microtubule and regulation of microtubule severing is critical for cellular processes such as ciliogenesis, morphogenesis, and meiosis and mitosis. Katanin is a conserved heterodimeric ATPase that severs and disassembles microtubules, but the molecular determinants for regulation of microtubule severing by katanin remain poorly defined. Here we show that the non-catalytic domains of Drosophila katanin regulate its abundance and activity in living cells. Our data indicate that the microtubule-interacting and trafficking (MIT) domain and adjacent linker region of the Drosophila katanin catalytic subunit Kat60 cooperate to regulate microtubule severing in two distinct ways. First, the MIT domain and linker region of Kat60 decrease its abundance by enhancing its proteasome-dependent degradation. The Drosophila katanin regulatory subunit Kat80, which is required to stabilize Kat60 in cells, conversely reduces the proteasome-dependent degradation of Kat60. Second, the MIT domain and linker region of Kat60 augment its microtubule-disassembly activity by enhancing its association with microtubules. On the basis of our data, we propose that the non-catalytic domains of Drosophila katanin serve as the principal sites of integration of regulatory inputs, thereby controlling its ability to sever and disassemble microtubules.

The Fog signaling pathway: insights into signaling in morphogenesis.

Epithelia form the building blocks of many tissue and organ types. Epithelial cells often form a contiguous 2-dimensional sheet that is held together by strong adhesions. The mechanical properties conferred by these adhesions allow the cells to undergo dramatic three-dimensional morphogenetic movements while maintaining cell-cell contacts during embryogenesis and post-embryonic development. The Drosophila Folded gastrulation pathway triggers epithelial cell shape changes that drive gastrulation and tissue folding and is one of the most extensively studied examples of epithelial morphogenesis. This pathway has yielded key insights into the signaling mechanisms and cellular machinery involved in epithelial remodeling. In this review, we discuss principles of morphogenesis and signaling that have been discovered through genetic and cell biological examination of this pathway. We also consider various regulatory mechanisms and the system׳s relevance to mammalian development. We propose future directions that will continue to broaden our knowledge of morphogenesis across taxa.

Excess centrosomes disrupt endothelial cell migration via centrosome scattering.

Supernumerary centrosomes contribute to spindle defects and aneuploidy at mitosis, but the effects of excess centrosomes during interphase are poorly understood. In this paper, we show that interphase endothelial cells with even one extra centrosome exhibit a cascade of defects, resulting in disrupted cell migration and abnormal blood vessel sprouting. Endothelial cells with supernumerary centrosomes had increased centrosome scattering and reduced microtubule (MT) nucleation capacity that correlated with decreased Golgi integrity and randomized vesicle trafficking, and ablation of excess centrosomes partially rescued these parameters. Mechanistically, tumor endothelial cells with supernumerary centrosomes had less centrosome-localized γ-tubulin, and Plk1 blockade prevented MT growth, whereas overexpression rescued centrosome γ-tubulin levels and centrosome dynamics. These data support a model whereby centrosome-MT interactions during interphase are important for centrosome clustering and cell polarity and further suggest that disruption of interphase cell behavior by supernumerary centrosomes contributes to pathology independent of mitotic effects.

The XMAP215 family drives microtubule polymerization using a structurally diverse TOG array.

XMAP215 family members are potent microtubule (MT) polymerases, with mutants displaying reduced MT growth rates and aberrant spindle morphologies. XMAP215 proteins contain arrayed tumor overexpressed gene (TOG) domains that bind tubulin. Whether these TOG domains are architecturally equivalent is unknown. Here we present crystal structures of TOG4 from Drosophila Msps and human ch-TOG. These TOG4 structures architecturally depart from the structures of TOG domains 1 and 2, revealing a conserved domain bend that predicts a novel engagement with α-tubulin. In vitro assays show differential tubulin-binding affinities across the TOG array, as well as differential effects on MT polymerization. We used Drosophila S2 cells depleted of endogenous Msps to assess the importance of individual TOG domains. Whereas a TOG1-4 array largely rescues MT polymerization rates, mutating tubulin-binding determinants in any single TOG domain dramatically reduces rescue activity. Our work highlights the structurally diverse yet positionally conserved TOG array that drives MT polymerization.

Short-circuiting microtubule plus and minus end proteins in spindle positioning.

Proteins residing at the plus and minus ends of microtubules have been thought not to communicate with each other, but recent findings on bona fide nucleation factors also regulating microtubule dynamics have challenged this notion. New work by Bouissou et al (2014) in The EMBO Journal now reveals that interplay between the nucleation factor γ­TuRC and the plus­end tracking protein EB1 controls mitotic spindle positioning by affecting the stability and dynamics of astral microtubules.

Gα12 structural determinants of Hsp90 interaction are necessary for serum response element-mediated transcriptional activation.

The G12/13 class of heterotrimeric G proteins, comprising the α-subunits Gα12 and Gα13, regulates multiple aspects of cellular behavior, including proliferation and cytoskeletal rearrangements. Although guanine nucleotide exchange factors for the monomeric G protein Rho (RhoGEFs) are well characterized as effectors of this G protein class, a variety of other downstream targets has been reported. To identify Gα12 determinants that mediate specific protein interactions, we used a structural and evolutionary comparison between the G12/13, Gs, Gi, and Gq classes to identify "class-distinctive" residues in Gα12 and Gα13. Mutation of these residues in Gα12 to their deduced ancestral forms revealed a subset necessary for activation of serum response element (SRE)-mediated transcription, a G12/13-stimulated pathway implicated in cell proliferative signaling. Unexpectedly, this subset of Gα12 mutants showed impaired binding to heat-shock protein 90 (Hsp90) while retaining binding to RhoGEFs. Corresponding mutants of Gα13 exhibited robust SRE activation, suggesting a Gα12-specific mechanism, and inhibition of Hsp90 by geldanamycin or small interfering RNA-mediated lowering of Hsp90 levels resulted in greater downregulation of Gα12 than Gα13 signaling in SRE activation experiments. Furthermore, the Drosophila G12/13 homolog Concertina was unable to signal to SRE in mammalian cells, and Gα12:Concertina chimeras revealed Gα12-specific determinants of SRE activation within the switch regions and a C-terminal region. These findings identify Gα12 determinants of SRE activation, implicate Gα12:Hsp90 interaction in this signaling mechanism, and illuminate structural features that arose during evolution of Gα12 and Gα13 to allow bifurcated mechanisms of signaling to a common cell proliferative pathway.

Regulation of epithelial morphogenesis by the G protein-coupled receptor mist and its ligand fog.

Epithelial morphogenesis is essential for shaping organs and tissues and for establishment of the three embryonic germ layers during gastrulation. Studies of gastrulation in Drosophila have provided insight into how epithelial morphogenesis is governed by developmental patterning mechanisms. We developed an assay to recapitulate morphogenetic shape changes in individual cultured cells and used RNA interference-based screening to identify Mist, a Drosophila G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor (GPCR) that transduces signals from the secreted ligand Folded gastrulation (Fog) in cultured cells. Mist functioned in Fog-dependent embryonic morphogenesis, and the transcription factor Snail regulated expression of mist in zygotes. Our data revealed how a cell fate transcriptional program acts through a ligand-GPCR pair to stimulate epithelial morphogenetic shape changes.

Drosophila Ric-8 interacts with the Gα12/13 subunit, Concertina, during activation of the Folded gastrulation pathway.

Heterotrimeric G proteins, composed of α, ß, and γ subunits, are activated by exchange of GDP for GTP on the Gα subunit. Canonically, Gα is stimulated by the guanine-nucleotide exchange factor (GEF) activity of ligand-bound G protein-coupled receptors. However, Gα subunits may also be activated in a noncanonical manner by members of the Ric-8 family, cytoplasmic proteins that also act as GEFs for Gα subunits. We used a signaling pathway active during Drosophila gastrulation as a model system to study Ric-8/Gα interactions. A component of this pathway, the Drosophila Gα12/13 subunit, Concertina (Cta), is necessary to trigger actomyosin contractility during gastrulation events. Ric-8 mutants exhibit similar gastrulation defects to Cta mutants. Here we use a novel tissue culture system to study a signaling pathway that controls cytoskeletal rearrangements necessary for cellular morphogenesis. We show that Ric-8 regulates this pathway through physical interaction with Cta and preferentially interacts with inactive Cta and directs its localization within the cell. We also use this system to conduct a structure-function analysis of Ric-8 and identify key residues required for both Cta interaction and cellular contractility.

The actin-microtubule cross-linking activity of Drosophila Short stop is regulated by intramolecular inhibition.

Actin and microtubule dynamics must be precisely coordinated during cell migration, mitosis, and morphogenesis--much of this coordination is mediated by proteins that physically bridge the two cytoskeletal networks. We have investigated the regulation of the Drosophila actin-microtubule cross-linker Short stop (Shot), a member of the spectraplakin family. Our data suggest that Shot's cytoskeletal cross-linking activity is regulated by an intramolecular inhibitory mechanism. In its inactive conformation, Shot adopts a "closed" conformation through interactions between its NH(2)-terminal actin-binding domain and COOH-terminal EF-hand-GAS2 domain. This inactive conformation is targeted to the growing microtubule plus end by EB1. On activation, Shot binds along the microtubule through its COOH-terminal GAS2 domain and binds to actin with its NH(2)-terminal tandem CH domains. We propose that this mechanism allows Shot to rapidly cross-link dynamic microtubules in response to localized activating signals at the cell cortex.