Alary muscles and thoracic alary-related muscles are atypical striated muscles involved in maintaining the position of internal organs.

Alary muscles (AMs) have been described as a component of the cardiac system in various arthropods. Lineage-related thoracic muscles (TARMs), linking the exoskeleton to specific gut regions, have recently been discovered in Drosophila Asymmetrical attachments of AMs and TARMs, to the exoskeleton on one side and internal organs on the other, suggested an architectural function in moving larvae. Here, we analysed the shape and sarcomeric organisation of AMs and TARMs, and imaged their atypical deformability in crawling larvae. We then selectively eliminated AMs and TARMs by targeted apoptosis. Elimination of AMs revealed that AMs are required for suspending the heart in proper intra-haemocelic position and for opening of the heart lumen, and that AMs constrain the curvature of the respiratory tracheal system during crawling; TARMs are required for proper positioning of visceral organs and efficient food transit. AM/TARM cardiac versus visceral attachment depends on Hox control, with visceral attachment being the ground state. TARMs and AMs are the first example of multinucleate striated muscles connecting the skeleton to the cardiac and visceral systems in bilaterians, with multiple physiological functions.

Small GTPases orchestrate cell-cell communication during collective cell movement.

Collective cell migration is a critical mechanism involved in cell movement during various physiological and pathological processes such as angiogenesis and metastasis formation. During collective movement, cells remain functionally connected and can coordinate individual cell behaviors to ensure efficient migration. A cell-cell communication process ensures this complex coordination. Although the mechanisms regulating cell-cell communication remain unclear, recent findings indicate that it is based on acto-myosin cytoskeleton tension transmission from cell to cell through adherens junctions. As for single cell migration, small GTPases of the Rho and Rab families have been shown to be critical regulators of collective motion. Here, we discuss our current understanding on how these small GTPases are themselves regulated and how they control cell-cell communication during collective migration. Moreover, we also shed light on the key role of cell-cell communication and RhoGTPases in the physiological context of endothelial cell migration during angiogenesis.

Non-autonomous role of Cdc42 in cell-cell communication during collective migration.

Collective cell migration is involved in numerous processes both physiological, such as embryonic development, and pathological such as metastasis. Compared to single cell migration, collective motion requires cell behaviour coordination through an as-yet poorly understood but critical cell-cell communication mechanism. Using Drosophila border cell migration, we show here that the small Rho GTPase Cdc42 regulates cell-cell communication. Indeed, we demonstrate that Cdc42 controls protrusion formation in a cell non-autonomous manner. Moreover, we found that the endocytic small GTPase Rab11, controls Cdc42 localisation to the periphery of migrating border cell clusters. Accordingly, over-expression of Cdc42 in border cells rescues the loss of Rab11 function. In addition, we showed that Cdc42 acts upstream of Moesin, a cytoskeletal regulator known to function downstream of rab11. Thus, our study positions Cdc42 as a new key player in cell-cell communication, acting downstream of Rab11.

Meiosis-specific stable binding of augmin to acentrosomal spindle poles promotes biased microtubule assembly in oocytes.

In the oocytes of many animals including humans, the meiotic spindle assembles without centrosomes. It is still unclear how multiple pathways contribute to spindle microtubule assembly, and whether they are regulated differently in mitosis and meiosis. Augmin is a γ-tubulin recruiting complex which "amplifies" spindle microtubules by generating new microtubules along existing ones in mitosis. Here we show that in Drosophila melanogaster oocytes Augmin is dispensable for chromatin-driven assembly of bulk spindle microtubules, but is required for full microtubule assembly near the poles. The level of Augmin accumulated at spindle poles is well correlated with the degree of chromosome congression. Fluorescence recovery after photobleaching shows that Augmin stably associates with the polar regions of the spindle in oocytes, unlike in mitotic cells where it transiently and uniformly associates with the metaphase spindle. This stable association is enhanced by γ-tubulin and the kinesin-14 Ncd. Therefore, we suggest that meiosis-specific regulation of Augmin compensates for the lack of centrosomes in oocytes by actively biasing sites of microtubule generation within the spindle.

Dgp71WD is required for the assembly of the acentrosomal Meiosis I spindle, and is not a general targeting factor for the γ-TuRC.

Dgp71WD/Nedd1 proteins are essential for mitotic spindle formation. In human cells, Nedd1 targets γ-tubulin to both centrosomes and spindles, but in other organisms the function of Dgp71WD/Nedd1 is less clear. In Drosophila cells, Dgp71WD plays a major part in targeting γ-tubulin to spindles, but not centrosomes, while in Xenopus egg extracts, Nedd1 acts as a more general microtubule (MT) organiser that can function independently of γ-tubulin. The interpretation of these studies, however, is complicated by the fact that some residual Dgp71WD/Nedd1 is likely present in the cells/extracts analysed. Here we generate a Dgp71WD null mutant lacking all but the last 12 nucleotides of coding sequence. The complete loss of Dgp71WD has no quantifiable effect on γ-tubulin or Centrosomin recruitment to the centrosome in larval brain cells. The recruitment of γ-tubulin to spindle MTs, however, is severely impaired, and spindle MT density is reduced in a manner that is indistinguishable from cells lacking Augmin or γ-TuRC function. In contrast, the absence of Dgp71WD leads to defects in the assembly of the acentrosomal female Meiosis I spindle that are more severe than those seen in Augmin or γ-TuRC mutants, indicating that Dgp71WD has additional functions that are independent of these complexes in oocytes. Moreover, the localisation of bicoid RNA during oogenesis, which requires γ-TuRC function, is unperturbed in Dgp71WD(120) mutants. Thus, Dgp71WD is not simply a general cofactor required for γ-TuRC and/or Augmin targeting, and it appears to have a crucial role independent of these complexes in the acentrosomal Meiosis I spindle.

Wac: a new Augmin subunit required for chromosome alignment but not for acentrosomal microtubule assembly in female meiosis.

The bipolar spindle forms without centrosomes naturally in female meiosis and by experimental manipulation in mitosis. Augmin is a recently discovered protein complex required for centrosome-independent microtubule generation within the spindle in Drosophila melanogaster cultured cells. Five subunits of Augmin have been identified so far, but neither their organization within the complex nor their role in developing organisms is known. In this study, we report a new Augmin subunit, wee Augmin component (Wac). Wac directly interacts with another Augmin subunit, Dgt2, via its coiled-coil domain. Wac depletion in cultured cells, especially without functional centrosomes, causes severe defects in spindle assembly. We found that a wac deletion mutant is viable but female sterile and shows only a mild impact on somatic mitosis. Unexpectedly, mutant female meiosis showed robust microtubule assembly of the acentrosomal spindle but frequent chromosome misalignment. For the first time, this study establishes the role of an Augmin subunit in developing organisms and provides an insight into the architecture of the complex.

Dual roles of Incenp crucial to the assembly of the acentrosomal metaphase spindle in female meiosis.

Spindle formation in female meiosis differs from mitosis in many animals, as it takes place independently of centrosomes, and the molecular requirements of this pathway remain to be understood. Here, we report two crucial roles of Incenp, an essential subunit of the chromosomal passenger complex (the Aurora B complex), in centrosome-independent spindle formation in Drosophila female meiosis. First, the initial assembly of spindle microtubules is drastically delayed in an incenp mutant. This clearly demonstrates, for the first time, a crucial role for Incenp in chromosome-driven spindle microtubule assembly in living oocytes. Additionally, Incenp is necessary to stabilise the equatorial region of the metaphase I spindle, in contrast to mitosis, where the equivalent function becomes prominent after anaphase onset. Our analysis suggests that Subito, a kinesin-6 protein, cooperates with Incenp for this latter function, but not in microtubule assembly. We propose that the two functions of Incenp are part of the mechanisms that compensate for the lack of centrosomes during meiotic spindle formation.

Drosophila melanogaster gamma-TuRC is dispensable for targeting gamma-tubulin to the centrosome and microtubule nucleation.

In metazoans, gamma-tubulin acts within two main complexes, gamma-tubulin small complexes (gamma-TuSCs) and gamma-tubulin ring complexes (gamma-TuRCs). In higher eukaryotes, it is assumed that microtubule nucleation at the centrosome depends on gamma-TuRCs, but the role of gamma-TuRC components remains undefined. For the first time, we analyzed the function of all four gamma-TuRC-specific subunits in Drosophila melanogaster: Dgrip75, Dgrip128, Dgrip163, and Dgp71WD. Grip-motif proteins, but not Dgp71WD, appear to be required for gamma-TuRC assembly. Individual depletion of gamma-TuRC components, in cultured cells and in vivo, induces mitotic delay and abnormal spindles. Surprisingly, gamma-TuSCs are recruited to the centrosomes. These defects are less severe than those resulting from the inhibition of gamma-TuSC components and do not appear critical for viability. Simultaneous cosilencing of all gamma-TuRC proteins leads to stronger phenotypes and partial recruitment of gamma-TuSC. In conclusion, gamma-TuRCs are required for assembly of fully functional spindles, but we suggest that gamma-TuSC could be targeted to the centrosomes, which is where basic microtubule assembly activities are maintained.

The Drosophila gamma-tubulin small complex subunit Dgrip84 is required for structural and functional integrity of the spindle apparatus.

Gamma-tubulin, a protein critical for microtubule assembly, functions within multiprotein complexes. However, little is known about the respective role of gamma-tubulin partners in metazoans. For the first time in a multicellular organism, we have investigated the function of Dgrip84, the Drosophila orthologue of the Saccharomyces cerevisiae gamma-tubulin-associated protein Spc97p. Mutant analysis shows that Dgrip84 is essential for viability. Its depletion promotes a moderate increase in the mitotic index, correlated with the appearance of monopolar or unpolarized spindles, impairment of centrosome maturation, and increase of polyploid nuclei. This in vivo study is strengthened by an RNA interference approach in cultured S2 cells. Electron microscopy analysis suggests that monopolar spindles might result from a failure of centrosome separation and an unusual microtubule assembly pathway via centriolar triplets. Moreover, we point to an involvement of Dgrip84 in the spindle checkpoint regulation and in the maintenance of interphase microtubule dynamics. Dgrip84 also seems essential for male meiosis, ensuring spindle bipolarity and correct completion of cytokinesis. These data sustain that Dgrip84 is required in some aspects of microtubule dynamics and organization both in interphase and mitosis. The nature of a minimal gamma-tubulin complex necessary for proper microtubule organization in the metazoans is discussed.